Dendritic Targeting Research Articles

In situ labeling of non-accommodating interneurons based on metabolic rates

Maintaining high frequency firing of narrow action potentials puts a large metabolic load on fast spiking (FS), perisomatic-inhibitory interneurons compared to their slow-spiking, dendrite targeting counterparts. Although the relationship of action potential (AP) firing and metabolism is firmly established, there is no single method to differentiate interneurons in situ based on their firing properties.In this study, we explore a novel strategy to easily identify the metabolically active FS cells among different classes of interneurons.We found that the oxidation of the fluorescent free radical marker 2,7-dichlorodihydrofluorescein (H2DCF) preferentially occurs in interneurons both in slice cultures and acute brain slices. Despite their morphological heterogeneity, almost all DCF-positive (DCF+) neurons belonged to the cluster of non-accommodating FS interneurons. Furthermore, all FS interneurons expressing parvalbumin (PV) both in slice cultures and in acute slices from tdTomato-PVCre transgenic mice were also DCF+. However, only half of the recorded DCF + cells were also PV+, indicating that H2DCF-oxidation occurs in different interneuron classes characterized by non-accomodating AP-firing. Comprehensively enhancing spontaneous neuronal activity led to mitochondrial oxidation of DCF in pyramidal cells as well as interneurons, suggesting that the apparent selectivity towards interneurons represents differences in the underlying metabolic load.While radical-scavenging, inhibition of APs or NO-synthesis, and iron chelation had no effect on the staining pattern, exposure to the complex-I inhibitor, rotenone, prevented interneuronal DCF accumulation. We conclude that H2DCF oxidation is independent of free radicals but correlates with the intensive oxidative energy metabolism and high mitochondrial mass in interneurons sharing the non-accommodating FS phenotype.

Multivalency Beats Complexity: A Study on the Cell Uptake of Carbohydrate Functionalized Nanocarriers to Dendritic Cells.

Herein, we report the synthesis of carbohydrate and glycodendron structures for dendritic cell targeting, which were subsequently bound to hydroxyethyl starch (HES) nanocapsules prepared by the inverse miniemulsion technique. The uptake of the carbohydrate-functionalized HES nanocapsules into immature human dendritic cells (hDCs) revealed a strong dependence on the used carbohydrate. A multivalent mannose-terminated dendron was found to be far superior in uptake compared to the structurally more complex oligosaccharides used.

Adaptable antigen matrix platforms for peptide vaccination strategies and T cell-mediated anti-tumor immunity

Injection of antigenic peptides has been widely used as a vaccine strategy to boost T cell immunity. However, the poor immunogenicity of single peptides can potentially be strengthened through modification of the tertiary structure and the selection of the accompanying adjuvant. Here, we generated antigenic peptides into non-linear trimers by solid phase peptide synthesis, thereby enhancing antigen presentation by dendritic cells to CD8+ T cells in vitro and in vivo. CD8+ T cells from mice vaccinated with trimers showed an KLRG1+ effector phenotype and were able to recognize and kill antigen-expressing tumor cells ex vivo. Importantly, trimers outperformed synthetic long peptide in terms of T cell response even when equal number of epitopes were used for immunization. To improve the synthesis of trimers containing difficult peptide sequences, we developed a novel small molecule that functions as conjugation platform for synthetic long peptides. This platform , termed Antigen MAtriX (AMAX) improved yield, purity and solubility of trimers over conventional solid phase synthesis strategies. AMAX outperformed synthetic long peptides in terms of both CD8+ and CD4+ T cell responses and allowed functionalization with DC-SIGN-binding carbohydrates for in vivo dendritic cell targeting strategies, boosting T cell responses even further. Moreover, we show that agonistic CD40 antibody combined with MF59 (AddaVax) emulsion synergistically improves the antigen-specific T cell response of the AMAX in vivo. Also, tumor-associated antigens and neo-antigens could be incorporated in AMAX for tumor-specific CD8+ T cell responses. Importantly, immunization with a mix of neoantigen AMAX could reduce tumor growth in a pre-clinical syngeneic mouse model. Hence, we provide pre-clinical support for the induction of effector CD8+ T cells through the adaptable AMAX platform as easy implementable peptidic vaccination strategy against any antigen of choice, including neoantigens for anti-tumor immunity.

MANα1-2MAN decorated liposomes enhance the immunogenicity induced by a DNA vaccine against BoHV-1.

New technologies in the field of vaccinology arise as a necessity for the treatment and control of many diseases. Whole virus inactivated vaccines and modified live virus ones used against Bovine Herpesvirus-1 (BoHV-1) infection have several disadvantages. Previous works on DNA vaccines against BoHV-1 have demonstrated the capability to induce humoral and cellular immune responses. Nevertheless, 'naked' DNA induces low immunogenic response. Thus, loading of antigen encoding DNA sequences in liposomal formulations targeting dendritic cell receptors could be a promising strategy to better activate these antigen-presenting cells (APC). In this work, a DNA-based vaccine encoding the truncated version of BoHV-1 glycoprotein D (pCIgD) was evaluated alone and encapsulated in a liposomal formulation containing LPS and decorated with MANα1-2MAN-PEG-DOPE (pCIgD-Man-L). The vaccinations were performed in mice and bovines. The results showed that the use of pCIgD-Man-L enhanced the immune response in both animal models. For humoral immunity, significant differences were achieved when total antibody titres and isotypes were assayed in sera. Regarding cellular immunity, a significant increase in the proliferative response against BoHV-1 was detected in animals vaccinated with pCIgD-Man-L when compared to the response induced in animals vaccinated with pCIgD. In addition, upregulation of CD40 molecules on the surface of bovine dendritic cells (DCs) was observed when cells were stimulated and activated with the vaccine formulations. When viral challenge was performed, bovines vaccinated with MANα1-2MAN-PEG-DOPE elicited better protection which was evidenced by a lower viral excretion. These results demonstrate that the dendritic cell targeting using MANα1-2MAN decorated liposomes can boost the immunogenicity resulting in a long-lasting immunity. Liposomes decorated with MANα1-2MAN-PEG-DOPE were tested for the first time as a DNA vaccine nanovehicle in cattle as a preventive treatment against BoHV-1. These results open new perspectives for the design of vaccines for the control of bovine rhinotracheitis.

Functional Role of Dendritic Cell Subsets in Cancer Progression and Clinical Implications.

Dendritic cells (DCs) constitute a complex network of cell subsets with common functions but also with many divergent aspects. All dendritic cell subsets share the ability to prime T cell response and to undergo a complex trafficking program related to their stage of maturation and function. For these reasons, dendritic cells are implicated in a large variety of both protective and detrimental immune responses, including a crucial role in promoting anti-tumor responses. Although cDC1s are the most potent subset in tumor antigen cross-presentation, they are not sufficient to induce full-strength anti-tumor cytotoxic T cell response and need close interaction and cooperativity with the other dendritic cell subsets, namely cDC2s and pDCs. This review will take into consideration different aspects of DC biology, including the functional role of dendritic cell subsets in both fostering and suppressing tumor growth, the mechanisms underlying their recruitment into the tumor microenvironment, as well as the prognostic value and the potentiality of dendritic cell therapeutic targeting. Understanding the specificity of dendritic cell subsets will allow to gain insights on role of these cells in pathological conditions and to design new selective promising therapeutic approaches.

Interplay between axonal Wnt5-Vang and dendritic Wnt5-Drl/Ryk signaling controls glomerular patterning in the Drosophila antennal lobe.

Despite the importance of dendritic targeting in neural circuit assembly, the mechanisms by which it is controlled still remain incompletely understood. We previously showed that in the developing Drosophila antennal lobe, the Wnt5 protein forms a gradient that directs the ~45˚ rotation of a cluster of projection neuron (PN) dendrites, including the adjacent DA1 and VA1d dendrites. We report here that the Van Gogh (Vang) transmembrane planar cell polarity (PCP) protein is required for the rotation of the DA1/VA1d dendritic pair. Cell type-specific rescue and mosaic analyses showed that Vang functions in the olfactory receptor neurons (ORNs), suggesting a codependence of ORN axonal and PN dendritic targeting. Loss of Vang suppressed the repulsion of the VA1d dendrites by Wnt5, indicating that Wnt5 signals through Vang to direct the rotation of the DA1 and VA1d glomeruli. We observed that the Derailed (Drl)/Ryk atypical receptor tyrosine kinase is also required for the rotation of the DA1/VA1d dendritic pair. Antibody staining showed that Drl/Ryk is much more highly expressed by the DA1 dendrites than the adjacent VA1d dendrites. Mosaic and epistatic analyses showed that Drl/Ryk specifically functions in the DA1 dendrites in which it antagonizes the Wnt5-Vang repulsion and mediates the migration of the DA1 glomerulus towards Wnt5. Thus, the nascent DA1 and VA1d glomeruli appear to exhibit Drl/Ryk-dependent biphasic responses to Wnt5. Our work shows that the final patterning of the fly olfactory map is the result of an interplay between ORN axons and PN dendrites, wherein converging pre- and postsynaptic processes contribute key Wnt5 signaling components, allowing Wnt5 to orient the rotation of nascent synapses through a PCP mechanism.

Paradoxical network excitation by glutamate release from VGluT3+ GABAergic interneurons.

In violation of Dale's principle several neuronal subtypes utilize more than one classical neurotransmitter. Molecular identification of vesicular glutamate transporter three and cholecystokinin expressing cortical interneurons (CCK+VGluT3+INTs) has prompted speculation of GABA/glutamate corelease from these cells for almost two decades despite a lack of direct evidence. We unequivocally demonstrate CCK+VGluT3+INT-mediated GABA/glutamate cotransmission onto principal cells in adult mice using paired recording and optogenetic approaches. Although under normal conditions, GABAergic inhibition dominates CCK+VGluT3+INT signaling, glutamatergic signaling becomes predominant when glutamate decarboxylase (GAD) function is compromised. CCK+VGluT3+INTs exhibit surprising anatomical diversity comprising subsets of all known dendrite targeting CCK+ interneurons in addition to the expected basket cells, and their extensive circuit innervation profoundly dampens circuit excitability under normal conditions. However, in contexts where the glutamatergic phenotype of CCK+VGluT3+INTs is amplified, they promote paradoxical network hyperexcitability which may be relevant to disorders involving GAD dysfunction such as schizophrenia or vitamin B6 deficiency.

Recombinant Lactococcus lactis co-expressing dendritic cell target peptide and E. tenella 3-1E protein: immune response and efficacy against homologous challenge

ABSTRACT In the present study, recombinant L. lactis NZ9000 displaying anchored dendritic cell-targeting peptide (DCpep) and E. tenella 3-1E protein was constructed. Chickens were orally immunized with the recombinant lactococci for three times at 2-week intervals. After three immunizations, chickens were challenged with E. tenella sporulated oocyst, and the immune response and protective efficacy were evaluated. The finding from animal experiments showed recombinant lactococci expressing anchored DCpep-3-1E protein elicited higher levels of 3-1E specific serum IgG and secretory IgA (sIgA) in caecal lavage, higher proportions of CD4+ and CD8α+ cells in peripheral blood, and higher mRNA expression levels of IL-2 and IFN-γ in the spleen compared to 3-1E-expressing lactococci, and hence offered obvious protective efficacy against homologous challenge. The present work demonstrated that cell surface-displayed target protein and immune enhancers in lactococci may be a promising approach to enhance immunity and immune efficacy against pathogen infection.

Cell-Surface Proteomic Profiling in the Fly Brain Uncovers Wiring Regulators

Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues and, thus, govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally resolved approach to profile cell-surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed global downregulation of wiring molecules and upregulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed invivo screen identified 20 cell-surface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally resolved in situ cell-surface proteomic profiling in discovering regulators of brain wiring.

Cell-Surface Proteomic Profiling in the Fly Brain Uncovers New Wiring Regulators

Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues, and thus govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally-resolved approach to profile cell-surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed a global down-regulation of wiring molecules and an up-regulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed in vivo screen identified 20 new cell-surface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally-resolved in situ cell-surface proteomic profiling in discovering new regulators of brain wiring.

Dendritic cell-based vaccine targeting aspartate-β-hydroxylas represents a promising therapeutic strategy for HCC.

Background: Dendritic cells (DCs)-mediated immunotherapy has been considered as a promising antitumor method. Aspartate-β-hydroxylase (AAH) is a potential immunotherapeutic target for hepatocellular carcinoma (HCC). Materials & methods: C57BL/6 mice were immunized by AAH-DCs vaccine constructed ex vivo. Killing tumor cells effect of active T cells induced by AAH-DCs vaccine on HCC cells were measured in vitro and vivo. The underlying mechanism was preliminarily investigated. Results: T cells response when activated by AAH-DCs vaccine showed a significant inhibition effect on HCC cells in vitro and in tumor-bearing mice models when compared with controls. Additionally, compared with the control group, increased expressions of Caspase8, Caspase 3 and Bax, and declined expression of Bcl-2 were observed in AAH-DCs vaccine group. Conclusion: AAH-DCs vaccine could stimulate T cell responses against HCC, which was possibly achieved via pro-apoptosis mechanism.

Neuronal BC RNA Transport Impairments Caused by Systemic Lupus Erythematosus Autoantibodies.

The etiology of the autoimmune disorder systemic lupus erythematosus (SLE) remains poorly understood. In neuropsychiatric SLE (NPSLE), autoimmune responses against neural self-antigens find expression in neurological and cognitive alterations. SLE autoantibodies often target nucleic acids, including RNAs and specifically RNA domains with higher-order structural content. We report that autoantibodies directed against neuronal regulatory brain cytoplasmic (BC) RNAs were generated in a subset of SLE patients. By contrast, anti-BC RNA autoantibodies (anti-BC abs) were not detected in sera from patients with autoimmune diseases other than SLE (e.g., rheumatoid arthritis or multiple sclerosis) or in sera from healthy subjects with no evidence of disease. SLE anti-BC abs belong to the IgG class of immunoglobulins and target both primate BC200 RNA and rodent BC1 RNA. They are specifically directed at architectural motifs in BC RNA 5' stem-loop domains that serve as dendritic targeting elements (DTEs). SLE anti-BC abs effectively compete with RNA transport factor heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) for DTE access and significantly diminish BC RNA delivery to synapto-dendritic sites of function. In vivo experiments with male BALB/c mice indicate that, upon lipopolysaccharide-induced opening of the blood-brain barrier, SLE anti-BC abs are taken up by CNS neurons where they significantly impede localization of endogenous BC1 RNA to synapto-dendritic domains. Lack of BC1 RNA causes phenotypic abnormalities including epileptogenic responses and cognitive dysfunction. The combined data indicate a role for anti-BC RNA autoimmunity in SLE and its neuropsychiatric manifestations.SIGNIFICANCE STATEMENT Although clinical manifestations of neuropsychiatric lupus are well recognized, the underlying molecular-cellular alterations have been difficult to determine. We report that sera of a subset of lupus patients contain autoantibodies directed at regulatory brain cytoplasmic (BC) RNAs. These antibodies, which we call anti-BC abs, target the BC RNA 5' domain noncanonical motif structures that specify dendritic delivery. Lupus anti-BC abs effectively compete with RNA transport factor heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) for access to BC RNAs. As a result, hnRNP A2 is displaced, and BC RNAs are impaired in their ability to reach synapto-dendritic sites of function. The results reveal an unexpected link between BC RNA autoantibody recognition and dendritic RNA targeting. Cellular RNA dysregulation may thus be a contributing factor in the pathogenesis of neuropsychiatric lupus.

Transsynaptic Fish-lips signaling prevents misconnections between nonsynaptic partner olfactory neurons

Our understanding of the mechanisms of neural circuit assembly is far from complete. Identification of wiring molecules with novel mechanisms of action will provide insights into how complex and heterogeneous neural circuits assemble during development. In the Drosophila olfactory system, 50 classes of olfactory receptor neurons (ORNs) make precise synaptic connections with 50 classes of partner projection neurons (PNs). Here, we performed an RNA interference screen for cell surface molecules and identified the leucine-rich repeat-containing transmembrane protein known as Fish-lips (Fili) as a novel wiring molecule in the assembly of the Drosophila olfactory circuit. Fili contributes to the precise axon and dendrite targeting of a small subset of ORN and PN classes, respectively. Cell-type-specific expression and genetic analyses suggest that Fili sends a transsynaptic repulsive signal to neurites of nonpartner classes that prevents their targeting to inappropriate glomeruli in the antennal lobe.

Mannosylated PEGylated-Polyethyleneimine as Efficient CpG Oligodeoxynucleotide Carriers for Efficient Dendritic Cell Targeting Delivery and Activation.

CpG ODN acts as a 'pathogen-associated' molecular pattern that is recognized by intracellular Toll-like receptor 9 and can induce a robust dendritic cells (DCs) activation to against various diseases. However, the CpG ODN is restricted with critical defects of easily enzymolysis and negligible phagocytosis. To overcome these issues, a simpler and competent nanocarrier of mannose modified PEGylated branched PEI25k (PEI-PEG-Man) was designed to achieve excellent DCsspecific delivery of CpG. Nanoparticles of PEI-PEG-Man encapsulating CpG (PEI-PEG-Man@CpG) possessed elevated gene loading capacity, biological stability and admirable anti-enzymolysis ability. PEI-PEG-Man@CpG could be selectively uptake by DCs through a receptor-mediated endocytosis, which generates a potent immunostimulatory activity on bone marrow derived dendritic cells (BMDCs) evidenced by significantly upregulation of the pro and anti-inflammatory cytokines (TNF-α, IL-6) and the co-stimulatory molecules (CD40, CD80, CD86, and MHC class II) on BMDCs in vitro. More importantly, the results of in vivo targeting assay showed that PEI-PEG-Man@CpG nanoparticles could remarkably boost CpG accumulation in lymph lodes upon subcutaneous administration in C57BL/6 mice, which facilitated maturation of DCs and productions of anti-inflammatory cytokines. Our results suggested that PEI-PEG-Man@CpG nanoparticles, in the future, might function as a powerful vector for ex vivo engineering to promote DC targeting and maturation, which enhance vaccine efficiency against cancer or infectious disease.

Dendritic Cell Targeting of Bovine Viral Diarrhea Virus E2 Protein Expressed by Lactobacillus casei Effectively Induces Antigen-Specific Immune Responses via Oral Vaccination.

Bovine viral diarrhea caused by bovine viral diarrhea virus (BVDV) is an important disease in cattle, resulting in significant economic losses to the cattle industry worldwide. In order to develop an effective vaccine against BVDV infection, we constructed a dendritic cell (DC)-targeting oral probiotic vaccine (pPG-E2-DCpep/LC W56) using Lactobacillus casei as antigen delivery carrier to express BVDV glycoprotein E2 fused with DC-targeting peptide, and the immunogenicity of orally administered probiotic vaccine was evaluated in mice model. Our results showed that after immunization with the probiotic vaccine, significantly levels of antigen-specific sera IgG and mucosal sIgA antibodies (p < 0.05) with BVDV-neutralizing activity were induced in vivo. Challenge experiment showed that pPG-E2-DCpep/LC W56 can provide effective immune protection against BVDV, and BVDV could be effectively cleared from the intestine of immunized mice post-challenge. Moreover, the pPG-E2-DCpep/LC W56 could efficiently activate DCs in the intestinal Peyer’s patches, and significantly levels of lymphoproliferative responses, Th1-associated IFN-γ, and Th2-associated IL-4 were observed in mice immunized with pPG-E2-DCpep/LC W56 (p < 0.01). Our results clearly demonstrate that the probiotic vaccine could efficiently induce anti-BVDV mucosal, humoral, and cellular immune responses via oral immunization, indicating a promising strategy for the development of oral vaccine against BVDV.

Predicted Markers of Overall Survival in Pancreatic Cancer Patients Receiving Dendritic Cell Vaccinations Targeting WT1

Background: We have developed a Wilms’ tumor 1 (WT1)-targeting dendritic cell (DC)-based cancer vaccine combined with standard chemotherapy for patients with advanced pancreatic ductal adenocarcinoma (PDA). Methods: We evaluated predictive markers of overall survival (OS) in PDA patients treated with multiple major histocompatibility complex class I/II-restricted, WT1 peptide-pulsed DC vaccinations (DC/WT1-I/II) in combination with chemotherapy. Throughout the entire period of immunochemotherapy, the plasma levels of soluble factors derived from granulocytes of 7 eligible PDA patients were examined. Moreover, systemic inflammatory response markers (neutrophil-to-lymphocyte ratio [NLR], monocyte-to-lymphocyte ratio [MLR], and granulocyte-to-lymphocyte ratio [GLR]) were assessed. In addition, cytoplasmic WT1 expression in PDA cells was examined. Results: Compared to the 4 non-super-responders (OS <1 year), the remaining 3 super-responders (OS ≥1 year) showed significantly decreased low plasma matrix metalloproteinase-9 levels throughout long-term therapy. The NLR, MLR, and GLR after 5 DC/WT1-I/II vaccinations and 3 cycles of gemcitabine were significantly lower in the super-responders than in the non-super-responders. Furthermore, the cytoplasmic WT1 expression in the PDA cells of super-responders was relatively weak compared to that in the PDA cells of non-super-responders. Conclusions: Prolonged low levels of a granulocyte-related systemic inflammatory response after the early period of therapy and low cytoplasmic WT1 expression in PDA cells may be markers predictive of OS in PDA patients receiving WT1-targeting immunochemotherapy.

Preliminary results of a phase I clinical trial using an autologous dendritic cell cancer vaccine targeting HER2 in patients with metastatic cancer or operated high-risk bladder cancer (NCT01730118).

2639 Background: We developed a HER2 targeting autologous dendritic cell (DC) vaccine transduced with an adenovirus expressing the extracellular and transmembrane domains of HER2 (AdHER2). In mice, the homologous vaccine cured virtually all mice with established or metastatic tumors. Protection was dependent on antibodies against HER2 that inhibited phosphorylation, but was ADCC independent. We translated these findings into a clinical trial. Methods: This is an open-label, phase I study in patients with 1) metastatic cancer that progressed after ≥ 1 standard therapies, or 2) history of high risk bladder cancer with definitive treatment, whose tumor is HER2 immunohistochemistry (IHC) score ≥ 1+ or FISH HER2/CEP17 ratio ≥ 1.8. Part 1 of the study enrolled patients naïve to HER2-directed therapies and Part 2 enrolled patients who progressed with ≥ 1 anti-HER2 therapy. Results: In Part 1, the lowest dose level (5E+6 viable DCs, N=7, 2 inevaluable) showed no benefit. At the second and third dose level (10E+6 and 20E+6; N=7 and N=4; 0 and 1 inevaluable in each), 1 CR (ovarian), 1 PR (stomach), and 3 SD (1 ovarian carcinosarcoma and 2 colon) were observed. Two bladder cancer patients who received vaccine as an adjuvant did not recur for +24 and +36 month each. In Part 2 (N=6, 2 inevaluable), 1 male breast cancer patient showed SD. Response assessed by Modified Immune Related Response Criteria is summarized in the Table. Injection-site reactions occurred in all patients and were self-limited. Echo, EKG and troponin follow up to 2 years showed no cardiac toxicity. Dose-expansion cohort (40E+6) is enrolling. Conclusions: We have translated a cancer vaccine from mice to a clinical trial. Preliminary results of a phase I trial of an autologous AdHER2 DC vaccine show potential clinical benefit in select patients with HER2 expressing tumors with no cardiac toxicity. Clinical trial information: NCT01730118. [Table: see text]

Development of a novel dendritic cell-based immunotherapy targeting cancer stem cells.

e14009 Background: Dendritic cells (DCs) are one of the central tools in cellular anti-tumor immunotherapy, being characterized by their capacity for acquiring and processing antigens and ability to produce strong antitumor immune responses. The production of clinical grade ex-vivo monocyte-derived DCs (Mo-DCs) is the most frequent approach for antitumor vaccines production. Recently, therapeutic resistance to radio/chemotherapy and disease recurrence was shown to be in part due to a small cancer stem cell (CSCs) population present in tumors. Methods: Here, we aim to target and eradicate CSCs by developing a novel DC-based immunotherapy vaccine for pancreatic and non-small cells lung cancer (NSCLC), comparing the loading of CSCs vs. classical tumor lysates. Results: CSCs from PANC-1 (pancreatic cancer) and A549 (NSCLC) cell lines were successfully isolated and characterized, overexpressing stem-like markers: NANOG, OCT4, SOX2 and CD133. CSCs resistance to Gemcitabine was also assessed. Before comparing the 2 types of vaccine loading, we also analyzed the impact of 3 GMP free-serum culture media on the phenotype and functional abilities of Mo-DCs. DCs cultured in X-VIVO 15 and AIM-V media show enhanced production of IL-12 and are able to induce a superior stimulation of T cells, mainly CTLs and Th1 subsets. By contrast, DCs cultured in DendriMACS are more prone to induce Treg polarization. Conclusions: Overall, our data demonstrate that blood monocytic precursors present considerable plasticity allowing a tailored differentiation of DCs just by changing the nutritive support. We also highlight the need of critically defining the culture medium to be used in DC cancer immunotherapy in order to attain desired cell characteristics and by consequent robust responses. Finally, our preliminary results indicate that loading DCs with CSCs antigens may be an effective strategy to target and destroy this resilient cancer cell population.

Oral Mucosal Immunization Recent Advancement and Exploit Dendritic Cell Targeting

Oral Mucosal Immunization Recent Advancement and Exploit Dendritic Cell Targeting

The Hunchback temporal transcription factor determines motor neuron axon and dendrite targeting in Drosophila.

The generation of neuronal diversity is essential for circuit formation and behavior. Morphological differences in sequentially born neurons could be due to intrinsic molecular identity specified by temporal transcription factors (henceforth called intrinsic temporal identity) or due to changing extrinsic cues. Here, we have used the Drosophila NB7-1 lineage to address this issue. NB7-1 generates the U1-U5 motor neurons sequentially; each has a distinct intrinsic temporal identity due to inheritance of different temporal transcription factors at its time of birth. We show that the U1-U5 neurons project axons sequentially, followed by sequential dendrite extension. We misexpressed the earliest temporal transcription factor, Hunchback, to create 'ectopic' U1 neurons with an early intrinsic temporal identity but later birth-order. These ectopic U1 neurons have axon muscle targeting and dendrite neuropil targeting that are consistent with U1 intrinsic temporal identity, rather than with their time of birth or differentiation. We conclude that intrinsic temporal identity plays a major role in establishing both motor axon muscle targeting and dendritic arbor targeting, which are required for proper motor circuit development.