WO2006008076A2 - Procedes et agents stimulant la reponse immunitaire - Google Patents

Procedes et agents stimulant la reponse immunitaire Download PDF

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WO2006008076A2
WO2006008076A2 PCT/EP2005/007690 EP2005007690W WO2006008076A2 WO 2006008076 A2 WO2006008076 A2 WO 2006008076A2 EP 2005007690 W EP2005007690 W EP 2005007690W WO 2006008076 A2 WO2006008076 A2 WO 2006008076A2
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jam
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mice
protein
agent
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Alberto Mantovani
Elisabetta Dejana
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Universita Degli Studi Di Milano
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Definitions

  • the present invention provides methods and agents stimulating the immune response in a mammalian organism through activation of dendritic cell motility and migration to lymph nodes.
  • agents inhibiting or blocking the transmembrane glycoprotein Junction Adhesion Molecule-A (JAM-A) expressed by dendritic cells are used as activators of specific immunity.
  • JAM-A Junctional Adhesion Molecule-A
  • JAM-A is a 32 kDa transmembrane glycoprotein present in endothelial, epithelial cells, in platelets and leukocytes. In the endothelium and several types of epithelia, JAM-A is located at intercellular junctions and codistributes with tight junction components (1). Other members of the JAM family (JAM-B and JAM-C) have been recently identified (2,3). These molecules present a more restricted cell distribution but the same localization at tight junctions (4-6).
  • the extracellular segment of JAM-A comprises two Ig-like domains, an amino terminal (VH- type) and a carboxyl terminal (C2-type) fold, respectively.
  • JAM-A may mediate homotypic cell to cell adhesion (7).
  • X-ray structure analysis suggests that JAM-A forms parallel and non-covalent homodimers, which might expose at the cell surface an adhesive interface for homophilic interactions (8). Due to its junctional distribution, JAM-A may control transendothelial migration of leukocytes.
  • An anti JAM-A mAb (BVI l) was able to reduce infiltration of both monocytes and polymorphonuclear cells in in vivo models of inflammation in mice (1,9).
  • JAM-A The mechanism of action of JAM-A in promoting cell transmigration remains undefined and it was suggested that it may have a role in binding leukocytes and in directing their transmigration through endothelial junctions.
  • JAM-A was found to link leukocyte integrin ⁇ L- ⁇ 2 (10) but homophilic binding of endothelial and leukocyte JAM-A may also occur. More recent data (11) show that endothelial JAM-A can reorganize at junctions during leukocyte transmigration creating a transient ring around transmigrating leukocytes. This suggests that JAM-A may actively promote the passage of the cells through the endothelial monolayer possibly creating a tunnel like structure at endothelial junctions.
  • DC Dendritic cells
  • the invention provides a method for increasing the motility and/or migration to lymph nodes of dendritic cells in mammals, preferably in humans, which comprises reducing or suppressing the activity of JAM-A protein expressed in these cells, alternatively by i) contacting the cells with an agent able to block or inhibit JAM-A, or ii) silencing the JAM-A gene in the dendritic cell.
  • the invention provides a method for stimulating the immune response in mammals, preferably in humans, which comprises administering to a subject in need of such treatment, an effective amount of an agent inhibiting or neutralizing JAM-A activity.
  • the invention provides a method for the vaccination, immunization or immunotherapy of mammals, preferably humans, which comprises administering to a subject in need of such a treatment, an effective amount of an agent inhibiting or neutralizing JAM-A activity.
  • an antigen such as a tumor antigen or an antigen associated with an infection disease
  • an agent inhibiting or neutralizing JAM-A activity for example an antibody to JAM-A protein or fragment thereof, or with dendritic cells bearing a silenced JAM-A gene optionally loaded with the specific antigen, to a subject affected by or susceptible to an infective or tumor disease, to stimulate an immune reaction.
  • the immunotherapeutic treatment according to the invention may comprise the steps of isolating dendritic cells from a subject, silencing JAM-A gene and reintroducing the cells into the subject.
  • the JAM- A-suppressed dendritic cells may be optionally loaded with the antigen before inoculation.
  • the JAM-A gene can be suppressed or silenced with genetic engineering or antisense technology.
  • the invention provides an immunostimulatory preparation containing an agent able to inhibit, block or neutralize JAM-A protein expressed by dendritic cells, or dendritic cells in which the JAM-A gene has been silenced.
  • agents inhibiting, blocking or neutralizing JAM-A protein expressed by dendritic cells are preferably selected among monoclonal or polyclonal antibodies to JAM-A protein or to fragments thereof.
  • the antibodies may be generated against either human or non-human JAM-A proteins and peptides. The generation of monoclonal antibodies with JAM-neutralizing activity is described in WO98/24897, which is herein incorporated by reference in its entirety.
  • JAM-A human and animal (mouse) amino acid sequences that can be used in the production of specific antibodies according to the present invention.
  • the monoclonal antibodies known as BV12, and, preferably, BVI l are utilized as JAM-A neutralizing or inhibiting agents.
  • the invention is directed to isolated dendritic cells and non-human animals bearing a silenced or suppressed JAM-A gene.
  • the JAM-A gene-suppressed non human animal can be used as a model for the study of the immune response.
  • the procedures for producing JAM-A -/- mice are described in the experimental section. DETAILED DESCRIPTION OF THE INVENTION
  • the invention is based on the identification of a novel, non-redundant, role of JAM-A in controlling DC motility, trafficking to lymph nodes and activation of specific immunity.
  • JAM-A -/- mice were generated and the functional behavior of DC in vitro and in vivo was studied.
  • JAM-A -/- DC showed a selective increase in random motility and in the capacity to transmigrate across lymphatic endothelial cells.
  • JAM-A -/- mice showed enhanced DC migration to lymph nodes, which was not observed in mice with endothelium restricted deficiency of the protein. Furthermore, increased DC migration to lymph nodes was associated with enhanced contact hypersensitivity.
  • JAM-A -/-mice were produced by generating a floxed JAM-A allele and by crossing these animals with CAG-Cre mice (13).
  • a targeting vector was designed such that a LoxP site and a floxed pGK neo resistance cassette were inserted into the 5' UTR region and into the intron 1 of JAM-A gene respectively ( Figure Ia).
  • Interbreeding of heterozygous mice for the JAM-A minus allele generated heterozygous and homozygous offspring in the correct Mendelian ratios. DNA isolated from tail biopsies were used for genotyping the animals.
  • JAM-A was undetectable both in vascular and lymphatic endothelial cells but retained its normal expression in epithelial cells.
  • the identification of Tie-2 Cre positive genotypes was done using the Tie-2 Cre primers as shown in Figure Ib. Then to distinguish between the heterozygous JAM-A flox/- and JAM-A flox/+ the primers TS379-512 were used.
  • JAM-A -/- DC showed normal chemotactic responsiveness to chemokines preferentially active on immature (CCL3) and mature (CCL 19) DC.
  • Transendothelial migration was then investigated.
  • JAM-A -/- and +/+ DC migration was equally low through blood microvascular endothelium (IGl 1).
  • MELC mouse lymphatic endothelial cells
  • JAM-A -/- DC showed a significant increase in transendothelial migration compared to JAM-A +/+ cells in both apical-to-basal and basal-to-apical direction.
  • JAM-A /- DC showed a marked increased in random motility as compared to JAM-A +/+ cells ( Figure 5c).
  • a JAM-A blocking mAb was added to JAM-A +/+ mDC and iDC we observed a significant increase in random motility: the area covered by migrating cells in a typical experiment was 112.11 ⁇ 16.56 ⁇ m 2 (mean ⁇ SEM) for controls and 337.31 ⁇ 10.56 in BVI l treated iDC (20 ⁇ g/ml); 104.96 ⁇ 13.96 and 598.27 ⁇ 17.36 ⁇ m 2 in controls and BVl 1 treated mDC respectively.
  • the FITC painting, contact sensitization assay was used to assess cutaneous DC migration in JAM-A -/mice.
  • FITC was applied to the skin of JAM-A +/+ and JAM-A -/- mice and the number of FITC/CDl lc double positive cells recovered from the draining lymph nodes was evaluated by FACS analysis.
  • Figure 7a the appearance of FITC + /CDl lc + cells was significantly increased in JAM-A -/- mice compared to JAM-A +/+.
  • the FITC painting assay was employed with Tie-2 Cre JAM-A -/- mice. In these animals, in contrast to the CAG-Cre JAM-A -/- mice, there was no significant increase but even a small but consistent decrease in DC migration to lymph nodes in comparison to JAM-A +/+ mice ( Figure 7b).
  • JAM-A -/- mice were studied in a contact hypersensitivity (CHS) model with oxazolone as sensitizing agent (see Methods).
  • Figure 8a shows the response of JAM-A +/+ and JAM-A -/- mice sensitized on day 0 and challenged 5 days later.
  • JAM-A -/- mice displayed an increased ear swelling compared to JAM-A +/+ mice at peak time (24 hours), which persisted up to 96 hours.
  • JAM-A expression was evaluated in lymph node and skin sections of JAM-A +/+ mice by immunohistochemistry.
  • JAM-A was strongly expressed on sinus macrophages (Figure 9b, asterisk) and in the paracortex on endothelial cells of high endothelial venules ( Figure 9b, c, arrowheads) and DC ( Figure 9c, arrow). No reactivity was observed on lymphoid cells ( Figure 9b, c).
  • DC of superficial lymph nodes include resident and migratory subsets. The latter are represented primarily by the nodal counterpart of skin-derived Langerhans cells (LC). Due to its role in DC migration from the periphery to lymph nodes, JAM-A expression was evaluated in normal skin sections.
  • JAM-A +/+ cells were represented by keratinocytes of the pilar follicle, endothelium of dermal vessels and scattered dermal cells with stellate/dendritic morphology ( Figure 9, insert in d). JAM-A reactivity was absent in lymph nodes and cutaneous sections from JAM-A -/- mice. Skin sections were immunostained for anti-MHC-class II molecules to identify LC in the epidermis.
  • LoxP primers were used.
  • Tie-2 Cre and TS379-512 primers were used.
  • TS379-512 generate a 500 bp product for minus allele, an 800 bp for wild-type allele and
  • JAM-A and ZO-I were expressed in confluent cells at intercellular contacts.
  • Cells presented a typical endothelial morphology.
  • Immature DC Immature DC
  • mDC mature DC
  • Methods Immature DC
  • JAM-A expression on JAM-A +/+ and JAM-A -/- iDC Thin line
  • mDC mDC
  • Dotted lines correspond to staining with an irrelevant mAb of an identical isotype.
  • mice 24 h after FITC-skin painting. Results are means ⁇ SEM of one representative experiments (8 mice per group) out of three performed.
  • CN cortical lymphoid nodules
  • PC expanded paracortical area
  • IVS-class II molecules insert in a
  • b Expression of JAM-A in lymph node section from JAM-A +/+ mouse is evident in sinus macrophages (asterisk), high endothelial venules (b and c, arrow-heads) and DC in the paracortex (c, black arrow).
  • Sections from an inguinal lymph node obtained after FITC skin painting display green dotted FITC + cells in the paracortex which coexpressed JAM-A (f, arrow) and Langerin (g, arrow). MHC-class H + and JAM-A + cells were detected by immunoperoxidase technique (insert in a, b, c, d).
  • Immunofluorescence staining was used to detect JAM-A + cells (red cells in f), MHCclass H + LC (red cells in e), Langerin "1" LC (red cells, insert in e; g).
  • a LoxP site is into the 5 ' UTR region of Exon 1 and a pGK neo resistance cassette flanked by two other LoxP sites is in opposite orientation into the intron 1.
  • JAM-A -/- mice were generated by crossing JAM-A Flox/Flox mice with CAG-Cre animals (13).
  • CAG Cre alleles the primers CAG Cre-F (5 '-CCAAAATTTGCCTGCATTACCGGTCGATGC- 3') and CAG Cre-R (5 '-AGCGCCGTAAATCAATCGATGAGTTGCTTC-S ') were used in PCR generating an 800 bp product.
  • LoxP primers TS447 (5'-CGTATAATGTATGCTATACGAAG-S ') and the antisense TS444 (5 '-GAGGTAGGGTCACAGATCACC-S ') were used (350 bp product in the presence of flox allele).
  • Endothelial JAM-A -/- mice were obtained as previously described (14).
  • the F-Tie-2 Cre (5'-CCCTGTGCTCAGACAGAAATGAGA-S') and the antisense primer Cre minus (5'-CCAGCAGGCGCACCATTGCCCCTG-S') were used in PCR generating a 500 bp product.
  • the Tie-2 Cre transgenic mice were a kind gift of Dr. Yanagisawa (University of Texas Medical Center, Dallas, TX) (16).
  • MELC Mouse lymphatic endothelial cells
  • VEGFR-3 Santa Cruz Biotechnology, Inc.
  • podoplanin a kind gift of Dr. Dontscho Kerjaschki; University of Vienna, Vienna, Austria
  • junction markers such as JAMA or ZO-I (Zymed Laboratories) (see Supplemental Figure 1 and Table 1).
  • Other antibodies used to characterize the cells are LYVE (a kind gift of Dr. E. Ruoslahti, Cancer Research Center, The Burnham Institute, La Jolla, CA and of Dr. K.
  • Cells were characterized in terms of membrane phenotype (expression of DEC205, ATCC HB-290; CD86, ATCC HB-253; anti-mouse I-A [Class II], clone 2G9 and anti-mouse CD80, clone IGlO from BD-Pharmingen San Diego, CA), pinocytosis and mixed lymphocyte response (MLR) on day 9 (40).
  • DC were cultured with TNF ⁇ (20 ng/ml) for the last 24 hours of culture.
  • rat anti mouse antibodies used to characterize DC or secondary lymphoid tissues were: CD 18 (ATCC GAME-46), CD3 (ATCC 145-2C11), CD19 (ATCC 1D3), APC and FITC-conjugated CDl Ib (ATCC Ml/70), PE-conjugated hamster anti-mouse CDl Ic (ATCC HL3), PerCP- Cy5.5-conjugated Gr-I (ATCC RB6-8C5), FITC-conjugated CD45/B220 (ATCC RA3-6B2), APC-conjugated CD8 ⁇ (clone 53-6.7) from BD-Pharmingen (San Diego, CA) and CDl Ia (ATCC Tib 217), L-selectin (ATCC HB- 132), F4-80 (ATCC HB 198) from ATCC hybridomas.
  • CD 18 ATCC GAME-46
  • CD3 ATCC 145-2C11
  • CD19 ATCC 1D3
  • Frozen lymph nodes were fixed in acetone (10 minutes at -20 0 C) and double stained with rat anti mouse JAM-A (BV 12) and rabbit anti mouse LYVE, over night at 4°C, followed by Cy3 donkey anti-rat and FITC goat anti-rabbit antibodies (Jackson). Fluorescence images of immunostained tissues were analyzed with Leica DMR fluorescence microscope and images recorded with a Hamamatsu 3CCD camera before processing through Adobe Photoshop for Macintosh. Thin tissue sections from lymph nodes were obtained and stained with haematoxylin/eosin for morphological evaluation.
  • lymph nodes and small fragments of mouse ears were embedded and frozen in Tissue Tec OCT. 3 ⁇ m thin sections were air-dried overnight, fixed in acetone (10 minutes) and incubated with 10% normal rabbit serum. Sections were then incubated with different primary antibodies including rat anti-mouse JAM-A (BV 12), MHC-class-II (clone 2G9, BD Pharmingen), Langerin (a king gift of Dr. G. Trinchieri, Shering Plough, Dardilly, France).
  • MHC-class II was performed by evaluating 30 high power fields (magnification 400x) obtained from non-consecutive ear sections. LC numbers were determined by counting MHCclass II positive cells with recognizable stellate morphology and normalized for the number of basal keratinocytes; data are expressed as mean number of LC/100 keratinocytes (41). Cytokines
  • Human CCL3 and CCL19 were from Peprotech Inc. (Rocky Hill, NJ).
  • Mouse granulocyte-macrophage-colony-stimulating factor (GM-CSF) was from Sandoz (Basel, Switzerland) and murine TNF- ⁇ was a kind gift from Dr. P. Vandenabeele (Gent University, Belgium).
  • Human Flt3 ligand was a generous gift from Immunex (Seattle, WA). Cytokines were endotoxin free as assessed by Limulus amebocyte assay (BioWhittaker Inc., Walkersville. MD). Chemotaxis and transmigration assays
  • Chemotaxis and trans-endothelial migration were performed in polycarbonate transwell inserts (5 ⁇ m pore, Corning, Costar, Cambridge, MA, USA) as previously described (40), with minor modifications.
  • 5I Cr-labeled DC (15xlO 4 /well in 0.1 ml for chemotaxis and 5xlO 4 /well in 0.1 ml for transmigration) were seeded in the upper compartment and chemoattractants were placed in the lower compartment. After 90 minutes of incubation at 37 0 C, the radioactivity present in the lower compartment was evaluated.
  • Chemokines were used at the optimal concentration of 100 ng/ml.
  • Results are reported as percentage of input, as in the following formula: (cpm in the lower compartment / cpm of the input) x 100.
  • the lymphatic endothelial cells (MELC) (38) or the microvascular mouse endothelial cell line IGl 1 (40) were grown as monolayer on gelatin- or fibronectin -coated insert; labeled DC were applied on the monolayers.
  • the MELC were grown on the upper or lower side of the filter.
  • the assay was performed as described (42). Briefly cells were plated onto glass cover slips previously coated with colloidal gold and incubated for 24 hours at 37°C. Then cells were fixed with 3% paraformaldehyde and observed by light microscopy. Pictures of individual cells were analyzed with the NIH ImageTM software for measuring the particle-free area and the number of tracks produced by each cell. In some experiments bone marrow-derived DC from JAM-A +/+ mice were incubated during the track assay with anti JAM-A mAb BVI l (1, 9) and anti PECAM mAb (MEC13.3) (37) as negative control (20 ⁇ g/ml).
  • FITC Fluorescein isothiocyanate Isomer I, Sigma, St. Louis, MO
  • BDH acetone- dibutylphthalate
  • Inguinal lymph nodes were obtained after 6 and 24 hours, mechanically disaggregated and treated with collagenase A (1 mg/ml, Boerhinger Mannheim, Indianapolis, IN) and DNase (0.4 mg/ml, Roche, Indianapolis, IN, USA) mixture for 30 min at 37°C and DNase alone at room temperature for 15 min. Cell suspensions were then stained with a PE-labelled hamster anti-mouse CDl Ic mAb (clone HL3, BD) and PE/FITC double-positive cells were analysed by FACScan.
  • CHS response was determined by measuring the degree of ear swelling of the antigen-painted ear compared to that of the vehicle-treated controlateral ear at different times after challenge (from 24 to 96 hours) using a dial thickness gauge (Mitutoyo, Cambridge, UK). The results were expressed as mean % of increased swelling calculated over vehicle-treated controlateral ear.
  • CHS was induced by inoculation in vivo of antigen- loaded DC (45). Cultured bone marrow derived DC were resuspended in HBSS without FCS containing 2,4-dinitrobenzene sulfonic acid (DNBS; ICN Biomedical Inc, Aurora, OH) 100 ⁇ g/ml and incubated at 37°C for 30 min.
  • DNBS 2,4-dinitrobenzene sulfonic acid
  • mice were challenged by the application of
  • mice 10 ⁇ l 2,4-dinitro-fluorobenzene (DNFB; Sigma, St. Louis) 0.2% or OXA 1% as negative control (in 4: 1 acetone:olive oil solution), on each side of right ear, while the left ear received the vehicle alone.
  • DNFB 2,4-dinitro-fluorobenzene
  • OXA OXA 1% as negative control (in 4: 1 acetone:olive oil solution), on each side of right ear, while the left ear received the vehicle alone.
  • Groups of mice injected with the same number of unmodified DC or not injected and challenged with the vehicle alone served as negative controls.
  • CHS response was determined as reported above for CHS to OXA.
  • mice are at least five mice. All experiments were performed at least three times. Statistical significance was evaluated using the two-tailed or the paired Student's t-test, as indicated.
  • Vascular endothelial junction-associated molecule a novel member of the immunoglobulin superfamily, is localized to intercellular boundaries of endothelial cells. J Biol Chem 275: 19139-19145.
  • JAM Junctional Adhesion Molecule
  • JAM-I is a ligand of the beta(2) integrin LFA-I involved in transendothelial migration of leukocytes. Nat Immunol 3: 151-158. 11. Ma, S., Shaw, S.K., Yang, L., Rao, R.M., Jones, T., Froio, R., Liu, Y., Nusrat, A., Parkos, C. A., and Luscinskas, F.W. 2003. Dynamics of Junctional Adhesion Molecule 1 (JAMl) during leukocyte transendothelial migration under flow in vitro. FASEB J (Abstr. 2712).
  • CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell 99:23-33.
  • the junctional multidomain protein AF-6 is a binding partner of the Rap IA GTPase and associates with the actin cytoskeletal regulator profilin. Proc Natl Acad Sd USA 97:9064-9069. 29. Cohen, A.R., Wood, D.F., Marfatia, S.M., Walther, Z., Chishti, A.H., and Anderson, J.M. 1998. Human CASK/LIN-2 binds syndecan-2 and protein 4.1 and localizes to the basolateral membrane of epithelial cells. J Cell Biol 142:129-138.
  • Vascular endothelial-cadherin is an important determinant of microvascular integrity in vivo. Proc Natl Acad Sci U S A 96:9815-9820.

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Abstract

L'invention concerne des agents supprimant l'activité de la protéine JAM-A ou inactivant le gène JAM-A dans des cellules dendritiques de mammifères, ainsi que l'utilisation de ces agents en tant qu'immunostimulants.
PCT/EP2005/007690 2004-07-16 2005-07-14 Procedes et agents stimulant la reponse immunitaire WO2006008076A2 (fr)

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US58837004P 2004-07-16 2004-07-16
US60/588,370 2004-07-16

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WO2006008076A2 true WO2006008076A2 (fr) 2006-01-26
WO2006008076A8 WO2006008076A8 (fr) 2006-03-30
WO2006008076A3 WO2006008076A3 (fr) 2006-07-27

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FR2909092A1 (fr) * 2006-11-24 2008-05-30 Pierre Fabre Medicament Sa Nouveaux anticorps anti-proliferation
JP2010509931A (ja) * 2006-11-24 2010-04-02 ピエール、ファーブル、メディカマン 新規抗増殖性化合物
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CN101535344B (zh) * 2006-11-24 2013-10-16 皮埃尔法布雷医药公司 新型抗增殖抗体

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