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ABSTRACT: Human plasmacytoid dendritic cells (pDCs) and myeloid dendritic cells (mDCs) are 2 types of antigen-presenting cells that exert complementary roles in innate immune responses. We demonstrated previously that in the presence of suboptimal stimulation or when only 1 dendritic cell type is directly stimulated, contact-dependent crosstalk between mDCs and pDCs leads to the activation of both cell types and thus provides them with the ability to induce an optimal T-cell response. The precise mechanism is currently unknown. Here we demonstrate that pDCs, unable to secrete soluble factors because of previous stimulation, induce optimal mDC maturation, indicating that resting immature mDCs are fully competent to respond to Toll-like receptor-9-engaged pDCs in the absence of soluble factors. Thus, we conclude that immature mDCs already express receptors recognized by ligands that are upregulated on the surface of activated pDCs. Intercellular adhesion molecule-1 upregulated by activated pDCs may play a role in a donor-dependent manner.
Human immunology 08/2011; 72(11):1018-21. · 2.55 Impact Factor
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ABSTRACT: Flow cytometry (FC) has been widely used in biological research; however, its use for vaccine characterization has been very limited. Here we describe the development of an FC method for the direct quantification of two Neisseria meningitidis vaccine antigens, in mono- and multivalent formulations, while still adsorbed on aluminum hydroxide (AH) suspension. The antibody-based method is specific and sensitive. Because FC allows microscopic particle examination, the entire aluminum suspension carrying adsorbed antigen(s) can be analyzed directly. In addition to determining antigen concentration and identity, the assay is able to determine the distribution of the antigens on AH. High correlation coefficients (r(2)) were routinely achieved for a broad range of antigen doses from 0 to 150 μg/dose. Traditional assays for quantitative and qualitative antigen characterization on AH particles involve either complete aluminum dissolution or antigen desorption from the adjuvant. Because our direct method uses the whole AH suspension, the cumbersome steps used by traditional methods are not required. Those steps are often inefficient in desorbing the antigens and in some cases can lead to protein denaturation. We believe that this novel FC-based assay could circumvent some of the complex and tedious antigen-adjuvant desorption methods.
Analytical Biochemistry 07/2011; 418(2):224-30. · 3.00 Impact Factor
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Carla Russo,
Ivan Cornella-Taracido,
Luisa Galli-Stampino,
Rishi Jain,
Edmund Harrington,
Yuko Isome,
Simona Tavarini, Chiara Sammicheli,
Sandra Nuti,
M Lamine Mbow,
Nicholas M Valiante,
John Tallarico,
Ennio De Gregorio,
Elisabetta Soldaini
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ABSTRACT: TLR7 and TLR8 are intracellular sensors activated by single-stranded RNA species generated during viral infections. Various synthetic small molecules can also activate TLR7 or TLR8 or both through an unknown mechanism. Notably, direct interaction between small molecules and TLR7 or TLR8 has never been shown. To shed light on how small molecule agonists target TLRs, we labeled 2 imidazoquinolines, resiquimod and imiquimod, and one adenine-based compound, SM360320, with 2 different fluorophores [5(6) carboxytetramethylrhodamine and Alexa Fluor 488] and monitored their intracellular localization in human plasmacytoid dendritic cells (pDCs). All fluorescent compounds induced the production of IFN-α, TNF-α, and IL-6 and the up-regulation of CD80 and CD86 by pDCs showing they retained TLR7-stimulating activity. Confocal imaging of pDCs showed that, similar to CpG-B, all compounds concentrated in the MHC class II loading compartment (MIIC), identified as lysosome-associated membrane protein 1(+), CD63, and HLA-DR(+) endosomes. Treatment of pDCs with bafilomycin A, an antagonist of the vacuolar-type proton ATPase controlling endosomal acidification, prevented the accumulation of small molecule TLR7 agonists, but not of CpG-B, in the MIIC. These results indicate that a pH-driven concentration of small molecule TLR7 agonists in the MIIC is required for pDC activation.
Blood 05/2011; 117(21):5683-91. · 9.90 Impact Factor
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Grazia Galli,
Duccio Medini,
Erica Borgogni,
Luisanna Zedda,
Monia Bardelli,
Carmine Malzone,
Sandra Nuti,
Simona Tavarini, Chiara Sammicheli,
Anne K Hilbert,
Volker Brauer,
Angelika Banzhoff,
Rino Rappuoli,
Giuseppe Del Giudice,
Flora Castellino
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ABSTRACT: Immune responses to vaccination are tested in clinical trials. This process usually requires years especially when immune memory and persistence are analyzed. Markers able to quickly predict the immune response would be very useful, particularly when dealing with emerging diseases that require a rapid response, such as avian influenza. To address this question we vaccinated healthy adults at days 1, 22, and 202 with plain or MF59-adjuvanted H5N1 subunit vaccines and tested both cell-mediated and antibody responses up to day 382. Only the MF59-H5N1 vaccine induced high titers of neutralizing antibodies, a large pool of memory H5N1-specific B lymphocytes, and H5-CD4(+) T cells broadly reactive with drifted H5. The CD4(+) response was dominated by IL-2(+) IFN-gamma(-) IL-13(-) T cells. Remarkably, a 3-fold increase in the frequency of virus-specific total CD4(+) T cells, measurable after 1 dose, accurately predicted the rise of neutralizing antibodies after booster immunization and their maintenance 6 months later. We suggest that CD4(+) T cell priming might be used as an early predictor of the immunogenicity of prepandemic vaccines.
Proceedings of the National Academy of Sciences 03/2009; 106(10):3877-82. · 9.68 Impact Factor
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Diego Piccioli, Chiara Sammicheli,
Simona Tavarini,
Sandra Nuti,
Elisabetta Frigimelica,
Andrea G O Manetti,
Annalisa Nuccitelli,
Susanna Aprea,
Sara Valentini,
Erica Borgogni,
Andreas Wack,
Nicholas M Valiante
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ABSTRACT: Dendritic cell (DC) populations play unique and essential roles in the detection of pathogens, but information on how different DC types work together is limited. In this study, 2 major DC populations of human blood, myeloid (mDCs) and plasmacytoid (pDCs), were cultured alone or together in the presence of pathogens or their products. We show that pDCs do not respond to whole bacteria when cultured alone, but mature in the presence of mDCs. Using purified stimuli, we dissect this cross-talk and demonstrate that mDCs and pDCs activate each other in response to specific induction of only one of the cell types. When stimuli for one or both populations are limited, they synergize to reach optimal activation. The cross-talk is limited to enhanced antigen presentation by the nonresponsive population with no detectable changes in the quantity and range of cytokines produced. We propose that each population can be a follower or leader in immune responses against pathogen infections, depending on their ability to respond to infectious agents. In addition, our results indicate that pDCs play a secondary role to induce immunity against human bacterial infections, which has implications for more efficient targeting of DC populations with improved vaccines and therapeutics.
Blood 02/2009; 113(18):4232-9. · 9.90 Impact Factor
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ABSTRACT: Costimuli provide supplementary signals required by naive T cells to become fully activated upon Ag encounter. Tetraspanins are a large family of transmembrane proteins that can costimulate T cells when engaged in vitro. In this study, we describe for the first time that coligation of the tetraspanins CD81, CD82, or CD9 with the costimulatory molecule CD28 in vitro leads to proliferation of naive T cells. When activated through this pathway, both CD4+ and CD8+ naive T cells differentiate into type 2 effector cells, which produce IL-4, IL-5, IL-13, and IL-10, together with IL-2 and TNF-alpha, but little to no IFN-gamma. These effector cells descend from precursors that display early and strong production of IL-4, STAT6 phosphorylation, and up-regulation of the transcription factor GATA-3, suggesting a direct skewing toward Th2 differentiation without a Th0 intermediate. The hepatitis C virus envelope protein E2 is the only ligand known for CD81. Therefore, we propose that this new type of Ag-independent T cell activation may occur in hepatitis C virus-infected individuals, contributing to liver inflammation, impaired type 1 immune responses, and recurrent flares of type 2 immunity associated with chronic infection.
The Journal of Immunology 08/2008; 181(1):174-85. · 5.79 Impact Factor
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ABSTRACT: Human blood contains 2 populations of dendritic cells (DCs): plasmacytoid and myeloid (mDC). mDCs are subdivided into 3 subsets using the surface markers CD16, CD1c, and BDCA-3. Their role as pathogen sentinels and adjuvant targets was tested by phenotypic and functional analysis. We show that mDC subsets are immature and express mRNA for most toll-like receptors (TLRs), except for TLR3 in CD16-mDCs. The most represented subsets, CD16- and CD1c-mDCs, are similarly responsive to all TLR agonists. Among 31 cytokines tested, both subsets produce CXCL8 (IL-8)/tumor necrosis factor-alpha (TNF-alpha)/IL-6/CCL3 (MIP-1 alpha)/CCL4 (MIP-1beta)/IL-1 beta. CXCL8 (IL-8) is the predominant cytokine produced by CD1c-mDCs on TLR engagement, whereas all other cytokines, particularly TNF-alpha, are secreted in 10-fold to 100-fold higher amounts by CD16-mDCs. CD16-mDCs cocultured with human umbilical vein endothelial cells induce a significantly higher production of CXCL10 (IP-10), granulocyte-macrophage colony-stimulating factor, and granulocyte colony-stimulating factor than CD1c-mDCs. In addition, interleukin-3 and type I interferons are stimuli specifically for DC maturation rather than cytokine secretion, whereas TNF-alpha is almost ineffective in inducing either function, suggesting a mechanism of T-cell-DC crosstalk and of rapid induction of antigen-presenting cell function during viral infection rather than inflammation. In conclusion, CD16-mDCs show strong proinflammatory activity, whereas CD1c-mDCs appear to be mainly inducers of chemotaxis.
Blood 07/2007; 109(12):5371-9. · 9.90 Impact Factor
