Massive-Scale RNA-Seq Analysis of Non Ribosomal Transcriptome in Human Trisomy 21

Hybridization- and tag-based technologies have been successfully used in Down syndrome to identify genes involved in various aspects of the pathogenesis. However, these technologies suffer from several limits and drawbacks and, to date, information about rare, even though relevant, RNA species such as long and small non-coding RNAs, is completely missing. Indeed, none of published works has still described the whole transcriptional landscape of Down syndrome. Although the recent advances in high-throughput RNA sequencing have revealed the complexity of transcriptomes, most of them rely on polyA enrichment protocols, able to detect only a small fraction of total RNA content. On the opposite end, massive-scale RNA sequencing on rRNA-depleted samples allows the survey of the complete set of coding and non-coding RNA species, now emerging as novel contributors to pathogenic mechanisms. Hence, in this work we analysed for the first time the complete transcriptome of human trisomic endothelial progenitor cells to an unprecedented level of resolution and sensitivity by RNA-sequencing. Our analysis allowed us to detect differential expression of even low expressed genes crucial for the pathogenesis, to disclose novel regions of active transcription outside yet annotated loci, and to investigate a plethora of non-polyadenilated long as well as short non coding RNAs. Novel splice isoforms for a large subset of crucial genes, and novel extended untranslated regions for known genes—possibly novel miRNA targets or regulatory sites for gene transcription—were also identified in this study. Coupling the rRNA depletion of samples, followed by high-throughput RNA-sequencing, to the easy availability of these cells renders this approach very feasible for transcriptome studies, offering the possibility of investigating in-depth blood-related pathological features of Down syndrome, as well as other genetic disorders

Vectorized delivery of alpha-Galactosylceramide and tumor antigen on filamentous bacteriophage fd induces protective immunity by enhancing tumor-specific T cell response

We have exploited the properties of filamentous bacteriophage fd to deliver immunologically active lipids together with antigenic peptides. Filamentous bacteriophages resemble for size, capability to be permeable to blood vessels, and high density antigen expression, a nature-made nanoparticle. In addition, their major coat protein pVIII, which is arranged to form a tubular shield surrounding the phage genome, has a high content of hydrophobic residues promoting lipid association. We conjugated bacteriophages to alpha-GalactosylCeramide (α-GalCer), a lipid antigen-stimulating invariant natural killer T (iNKT) cells and capable of inducing their anti-tumoral activities. We found that bacteriophage fd/α-GalCer conjugates could repeatedly stimulate iNKT cells in vitro and in vivo, without inducing iNKT anergy. Moreover, co-delivery of α-GalCer and a MHC class I restricted tumor-associated antigenic determinant to antigen-presenting cells via bacteriophages strongly boosted adaptive CD8+ T cell response and efficiently delayed tumor progression. Co-delivery of a tumor antigen and iNKT-stimulatory lipid on the surface of filamentous bacteriophages is a novel approach to potentiate adaptive anti-cancer immune responses, overcoming the current limitations in the use of free α-GalCer and may represent an attractive alternative to existing delivery methods, opening the path to a potential translational usage of this safe, inexpensive, and versatile tool

Induction of human NK cell-mediated cytotoxicity by CD40 triggering on antigen presenting cells

Engagement of CD40 on antigen presenting cells (APC) is central to the initiation of cell-mediated immune response. Here, we investigated the ability of CD40 ligation on APC to induce NK cell-mediated cytotoxicity in the human system and the mechanism(s) underlying this process. We showed that APC (consisting in adherent peripheral blood mononuclear cells) (PBMC), prestimulated with anti-CD40 monoclonal antibodies and co-cultured with autologous non-adherent PBMC for 5-9 days, induced CD3-/CD56+ NK cell-mediated cytotoxicity as well as CD3+/CD56+ T cell-mediated unrestricted cytotoxic activity. The generation of NK cell-mediated cytotoxicity was independent on cell-to-cell contact between CD40-triggered APC and NK cells. Moreover. we found that IL-12 did not play a role in NK cells induction by anti-CD40 priming, while IL-2 and IL-15 did play a role. Our results provide an insight into the mechanism by which NK cells are activated in peripheral blood and useful informations for therapeutic application of anti-CD40 antibodies. (C) 2003 Elsevier Science (USA). All rights reserved

Triggering DTH and CTL Activity by fd Filamentous Bacteriophages: Role of CD4+ T Cells in Memory Responses

The ability of fd bacteriophage particles to trigger different arms of the immune system has been previously shown by us with particular emphasis on the ability of phages to raise CTL responses in vitro and in vivo. Here we show that fd virions in the absence of adjuvants are able to evoke a DTH reaction mediated by antigen specific CD8+ T cells. In addition, we analyzed the induction of CTL responses in mice depleted of CD4+ T cells, and we observed that short-term secondary CTL responses were induced in the absence of CD4+ T cells while induction of long-term memory CTLs required the presence of CD4+ T lymphocytes. These results examine the cellular mechanism at the basis of fd efficiency and provide new elements to further validate the use of fd particles for eliciting and monitoring antigen-specific CTLs

The use of filamentous bacteriophage fd to deliver MAGE-A10 or MAGE-A3 HLA-A2-restricted peptides and to induce strong antitumor CTL responses

Delivery of tumor-associated antigen (TAA)-derived peptides in a high immunogenic form represents one of the key issues for effective peptide-based cancer vaccine development. Here, we report the ability of non-pathogenic filamentous bacteriophage fd virions to deliver HLA-A2-restricted MAGE-A10254-262- or MAGE-A3271-279-derived peptides and elicit potent specific CTL responses in vitro and in vivo. Interestingly, human anti-MAGE-A3271-279-specific CTLs were able to kill human MAGE-A3+ tumor cells, even if these cells naturally express a low amount of MAGE-A3271-279 peptide-HLA epitope surface complexes and are usually not recognized by CTLs generated by conventional stimulation procedures. MAGE-A3271-279-specific/CD8+ CTL clones were isolated from in vitro cultures and their high avidity for antigen recognition was assessed. Moreover, in vivo tumor protection assay showed that vaccination of humanized HHD (HLA-A2.1+/H2-Db+) transgenic mice with phage particles expressing MAGE-A3271-279-derived peptides hampered tumor growth. Overall, these data indicate that engineered filamentous bacteriophage virions increase substantially the immunogenicity of delivered TAA-derived peptides, thus representing a novel powerful system for the development of effective peptide-based cancer vaccines.L'articolo è disponibile sul sito dell'editore http://www.jimmunol.or

Antigen delivery by filamentous bacteriophage fd displaying an anti-DEC-205 single-chain variable fragment confers adjuvanticity by triggering a TLR9-mediated immune response

Filamentous bacteriophage fd particles delivering antigenic determinants via DEC-205 (fdsc-αDEC) represent a powerful delivery system that induces CD8(+) T-cell responses even when administered in the absence of adjuvants or maturation stimuli for dendritic cells. In order to investigate the mechanisms of this activity, RNA-Sequencing of fd-pulsed dendritic cells was performed. A significant differential expression of genes involved in innate immunity, co-stimulation and cytokine production was observed. In agreement with these findings, we demonstrate that induction of proinflammatory cytokines and type I interferon by fdsc-αDEC was MYD88 mediated and TLR9 dependent. We also found that fdsc-αDEC is delivered into LAMP-1-positive compartments and co-localizes with TLR9. Thus, phage particles containing a single-strand DNA genome rich in CpG motifs delivered via DEC-205 are able to intercept and trigger the active TLR9 innate immune receptor into late endosome/lysosomes and to enhance the immunogenicity of the displayed antigenic determinants. These findings make fd bacteriophage a valuable tool for immunization without administering exogenous adjuvants

Binding antibody responses in rabbits co-immunized with Env(V3)-E2 particles and DNA with and without LPS and IFA.

<p>Specific antibody responses against E2 (E2wt) (A), HIV-1 Env(V3) peptide (B) and Env(gp140) (C) determined by ELISA. Data shown are geometric mean values (+/− S.D.) from rabbits in Groups 6 (purple), 7 (black) and 8 (orange). Colored arrows at the top of the figure indicate the time of immunization for each group.</p

H2^d-Dextramer Staining and Intracellular Cytokine Staining of CD8⁺ T Cells from Immunized Mice.

a<p>ICS, intracellular cytokine staining;</p>b<p>SD, standard deviation;</p>c<p>Values are expressed as percentages (%) minus the average percentage of the cells with medium alone; LPS, lipopolysaccharide; –, not done.</p

Neutralizing antibody responses in rabbits co-immunized with Env(V3)-E2 particles and DNA with and without LPS and IFA.

<p>(<b>A–C</b>) NAbs against HIV-SF162 in sera from rabbits immunized with Env(V3)-E2 co-administered with Env(gp160) DNA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031464#pone-0031464-t001" target="_blank">Table 1</a>, Groups 6–8. Lines indicate NAb titers for individual rabbits in each group: (A) Group 6, LPS-negative Env(V3)-E2, plus IFA; (B) Group 7, LPS-negative, no IFA; (C) Group 8, LPS-positive, plus IFA. Arrows at the bottom on the figure indicate time and type of immunization. The dotted line represents the limit of detection of the assay (1∶16). (<b>D</b>) Comparison of the NAb area under the curve (AUC) between groups immunized following similar regimens and using same immunogens with (Groups 5 and 6) or without the addition of IFA (Group 7).</p

Neutralizing antibody responses in rabbits immunized with Env-E2 particles and DNA following different immunization regimes.

<p>(<b>A–E</b>) Lines indicate NAbs <i>versus</i> HIV-SF162 in each rabbit in Groups 1 (blue), 2 (red), 3 (green), 4 (golden) and 5 (purple). Arrows at the bottom of each graph indicate time and type of immunization: grey arrows, DNA vaccination; black arrows, Env-E2 VLPs; combined grey and black arrows, co-administration of DNA plus E2. (<b>F</b>) NAb titers during priming stages for three immunization regimens: (i) Env-E2 (Group 1, in blue), (ii) DNA (Group 2, in red) and (iii) co-administration of Env-E2 plus DNA (Groups 3, 4 and 5; in green). Bars are mean titer values (+S.D.) in each group. Asterisks denote statistical significance: ** P<0.01; * P<0.05. Dotted lines indicate the limit of detection of the assays.</p