JPH10513350A - Identification of DEC (dendritic and epithelial cells, 205 kDa), receptor with C-type lectin domain, nucleic acid encoding DEC, and uses thereof - Google Patents

Abstract

SUMMARY The present invention relates to the identification and characterization of receptors associated with antigen presentation in immune responses, endocytosis, and transepithelial transport. The identification of the receptor, its characterization as having 10 lectin binding domains, and demonstration of its role in the uptake and processing of oligosaccharides and oligosaccharide-modifying molecules, such as glycoproteins, has been shown to improve the immune response and It has important related problems in epithelial transport. The receptor, an integral membrane protein, referred to herein as "DEC", is found primarily in dendritic cells, but also in B cells, brain capillaries, bone marrow stromes, intestinal parenchyma and pulmonary airways. And dendritic cells in the T cell region of thymic cortical epithelium and peripheral lymphoid organs. The mouse and human counterparts of DEC have an apparent molecular weight of 205 kDa, the mouse counterpart has an isoelectric point at pH 7.5, and the carbohydrate is about 7 kDa of the total mass of mouse DEC. The present invention relates to the identification of additional ligands for the DEC-205 receptor, which can advantageously target dendritic cells and other cells having DEC-205. Targeting antigens for presentation by dendritic cells can confer tolerance when the dendritic cells are quiescent, or the dendritic cells can be activated by, for example, cytokines or lymphokines, such as colony stimulating factor (CSF) An immune stimulus (ie, vaccination) can be given when activated by the stimulus.

Description

DETAILED DESCRIPTION OF THE INVENTION Identification of DEC (dendritic and epithelial cells, 205 kDa), with C-type lectin domain Receptor, nucleic acid encoding DEC, and uses thereof   The work leading to the present invention was supported in part by the National Health Association Grant No. AI13013. the government The invention has certain rights.   Field of the invention   The present invention relates to antigen presentation in the immune response, endocytosis, and transepithelial transport. It concerns the identification and characterization of related receptors. Identification of receptors, 10 lek Characterization as having a tin binding domain, and oligosaccharides and oligos Glyco-modifying molecules, such as their role in glycoprotein uptake and processing Evidence has important implications for improving the immune response and transepithelial transport of molecules.   Background of the Invention   Dendritic cells are primarily responsible for capturing, processing, and presenting their antigens to T cells Is a unique class of leukocytes that is to perform (Steinman, 1991, Annu. Rev. Immu-n ol. 9: 271-96). Interactions between dendritic cells and specific T cells in the peripheral immune system are immune Leads to induction of response, while presentation by dendritic cells has a negative choice in the thymus (Tanaka et al., 1993, Eur. J. Immunol. 23: 2614-2621; Matzinger et al., 1989, Natu. re 338: 74-76). Like dendritic cells, thymic epithelial cells make T cells MHC-binding peptides. Thymic epithelial cells instead of inducing T cell activation or negative selection. Vesicles induce positive selection (Hugo et al., 1993, Immunol. Rev. 135: 133-35; Elli-ott. Imunol. Rev. 135: 215-25). Consistent with known requirements for interaction with T cells, dendritic Both cells and thymic epithelial cells promote cell-cell contact and activate T cells Express several mediating cell surface proteins (Steinman, 1991, Annu. Rev I rnmunol. 9: 271-96; Hugo et al., 1993, Irmunol. Rev. 135: 133-35; Elliott. Immunol. Rev. 135215-25). Additional for both dendritic cells and thymic epithelial cells The basic requirement is antigen uptake and processing, and any cell type can capture antigen. Or, it is not yet known to express receptors that are specialized for presentation.   Over 10 years since its initial isolation by Kraal et al. (J. Exp. Med. 163: 981) Monoclonal antibody NLDC-145 organizes mouse dendritic cells (DC) in various tissues (Kraal Crowley et al., 1989, Cell Immunol. 118: 108; Vremec et al., 1992, J.Exp.Med. 176: 47; Pollard and Lipscomb, 1990, J. Exp. Med. 172: 159; Soesa- tyo et al., 1990, Cell Tiss. Res. 259: 587; Austyn et al., 1994, J. Immunol. 152: 2401 Lu et al., 1994, J. Exp. Med. 179: 1823; and Breel et al., 1988, Immunol. 63: 657). Also, antigens bound by NLDC-145 are abundant in thymic cortical epithelium. However Found that cloning and characterization of the NLDC-145 antigen proved elusive. . One reason is that dendritic cell cDNA libraries are not easily prepared. Was. Dendritic cells themselves are rare, making their RNA extremely rare. In addition, Noclonal antibodies are usually expressed in an expression library, for example, a λgt-11 expression library. Is not an effective reagent for screening   Therefore, the antigen recognized by the monoclonal antibody NLDC-145 was cloned, There is a need in the art for characterization.   Utilizing the immunoregulatory ability of dendritic cells, for example, to induce tolerance or immunity There is a further need in the art for this.   Citation of a document in this specification indicates that such document is prior art to the present invention. It should not be regarded as admission.   Summary of the Invention   The present invention is found primarily in dendritic cells, but also in B cells, brain capillaries, bone marrow. Stroma, intestinal parenchyma and pulmonary airway epithelium, and thymic cortical epithelium and peripheral lymph Referred to herein as "DEC", which is found in dendritic cells in the T cell region of a systemic organ , Related to integral membrane proteins. In addition, trace amounts of this protein are found in the liver, heart Offal, And found in organs such as the kidney. The mouse and human counterparts of DEC are 20 It has an apparent molecular weight of 5 kDa, the mouse counterpart has an isoelectric point at pH 7.5 and is carbohydrated. Contain approximately 7 kDa of the total mass of mouse DEC. There are eight different carbohydrates, It is clear that it consists of two related (biantennary) N-linked glycans. And no O-linked glycans are present. Its proteins are mainly dendritic cells and thymus It is called DEC-205 because it is found in epithelial cells and has a molecular weight of 205 kDa. It is. In a particular embodiment, the invention relates to the isolation and cloning of human DEC. This is the carboxyl-terminal sequence RHRLHLAGFSSVRYAQGVNEDEIMLPSF HD (SEQ ID NO: 1) and is characterized by having full-length mouse DEC-205 Binds to rabbit polyclonal antibodies raised against the monoclonal antibody NLDC Characterized by not reacting with -145. In a more specific embodiment, DEC has the amino acid sequence shown in SEQ ID NO: 8. It is shown in SEQ ID NO: 7 Encoded nucleotide sequence. Cloning dec cDNA Another feature of DEC, based on deduced amino acid sequence information obtained from A unique coated pit-localized consensus domain in the cytoplasmic tail of parkin Or motif, which, as shown in FIG. It has regions of homology and differences between proteins. DEC-205 is faster with coated vesicles A small internalization agent that rapidly internalizes and binds binding substances to MHC class II, which is involved in antigen processing. It has further been discovered that delivery to multivesicular endosomal compartments resembling vesicles.   Therefore, the present invention, in its main aspect, provides additional DEC-205 receptor For the identification of ligands, including dendritic cells and those with DEC-205. Other cells may be advantageously targeted. Targeting antigens for presentation by dendritic cells Can provide tolerance when the dendritic cells are at rest, or The vesicle is, for example, a cytokine or lymphokine, such as a colony stimulating factor ( Provides immune stimulation (ie, vaccination) when activated by stimulation by CSF) be able to.   In addition, the presence of DEC in epithelial cells, From the outer surface to the lung airways, or from the basolateral surface of the intestinal epithelium to the lumen of the intestine, and In the transepithelial transport of molecules from the apical (luminal) surface of their epithelium to the lung or intestinal circulation Suggest an important role. Thus, the present invention provides a pharmacological agent having a ligand. For pulmonary or intestinal infections by targeting to DEC For example, parenteral delivery of antibiotics, and aerosol application and pulmonary inhalation, or Or systemic delivery of pharmacological agents by ingestion and absorption by the intestine.   In yet another aspect, the present invention provides a blood-brain barrier with DECs located in brain capillaries. Provides targeting of pharmacological agents across the gate.   Thus, in a preferred aspect, the invention provides at least one DEC lectin Contacting a protein containing a domain with a candidate ligand, the DEC lectin domain Of DEC ligands characterized by detecting the binding of candidate ligands by HPLC Provide a way. The binding of the candidate ligand to the DEC lectin domain is Indicates that complement is a ligand for DEC. In a preferred aspect, the ligand is A saccharide, which binds to one or more lectin domains of the DEC.   According to one aspect of the present invention, the method comprises at least one DEC lectin domain. Is expressed by the cell as an integral membrane protein, and candidate ligands are Labeled so that the binding of the candidate ligand by the DEC lectin domain is Is detected by detecting the association of the label with. In another embodiment, Proteins containing at least one DEC lectin domain are solubilized and Is irreversibly associated with the solid support, resulting in a DEC lectin domain. Binding of candidate ligands to proteins detects protein binding by the solid support More detected. In yet another embodiment, at least one DEC lectin The protein containing the domain is irreversibly associated with the solid support, and the candidate ligand is labeled. As a result, the binding of the candidate ligand by the DEC lectin domain is It is detected by detecting the association of the label with the body. In one embodiment, less Proteins containing at least one DEC lectin domain are truncated DEC It is protein. In another embodiment, at least one DEC lectin domain The protein containing the in is a full-length DEC protein.   The present invention advantageously provides nucleic acids encoding at least a portion of a DEC protein You. Thus, the present invention relates to DEC proteins, or Provides expression of the truncated fragment, which identifies the DEC ligand Can be used for Further, the nucleic acids of the invention comprise at least 15 base pairs, thus The nucleic acids of the invention are useful probes for detecting the expression of DEC mRNA, PCR primers for reverse transcriptase polymerase chain reaction (RT-PCR), or DE Probes for cloning C and, for example, libraries or cells Provide a probe for the presence of DEC cDNA or genomic DNA in the DNA. In a preferred embodiment, the nucleic acid encodes a human DEC protein. below In a specific embodiment, a nucleic acid encoding human DEC is provided.   Furthermore, the present invention provides an expression vector comprising a nucleic acid encoding DEC, Acid is at least the lectin domain of the DEC operatively associated with the expression control sequence Is a DNA molecule that encodes In yet another aspect, the present invention relates to an expression vector The present invention provides a recombinant host cell comprising In various embodiments, the host cell is a host cell. Chinese hamster ovary cells, African green monkey COS cells, Gin-Darby canine kidney cells and NIH-3T3 fibroblasts Mammalian cells.   Further, the present invention relates to antibodies reactive with human DEC-205 protein, especially monoclonal antibodies. Antibodies and polyclonal antibodies are provided.   As mentioned above, the present invention advantageously provides for the identification of DEC ligands, and Can adhere to cells that have DEC in vitro or in vivo; The molecules to be combined can be targeted. Target cells expressing DEC in vivo The ability to target, for example, specifically targets thymus, small intestine, and lung dendritic cells, epithelial cells This has important implications from targeting expectations. Thus, naturally, the present invention provides pulmonary circulation. T of dendritic cells and lymphoid organs in the annulus, intestinal circulatory system, lung airways, lumen of small intestine, skin Physician containing a molecule that targets a tissue selected from the group consisting of cell region, thymus, and brain A drug composition, the molecule of which is conjugated to a DEC-ligand. DEC-ligands are carbohydrate and anti-DEC antibodies that bind DEC, and pharmaceutically acceptable It is preferable to be selected from the group consisting of carriers to be used. In particular embodiments, The molecules are from anticancer, antiviral, antibiotic, antiparasitic, and anti-inflammatory drugs. Selected from the group.   In another aspect of the invention relating to targeting, the DEC-ligand Dendritic cells, thymic epithelial cells, lung epithelial cells, on the small intestine containing the combined DNA vector For gene transfer into cells selected from the group consisting of skin cells and brain capillary cells Wherein the DEC-ligand is a carbohydrate that binds DEC. And an anti-DEC antibody. In a specific embodiment, the DNA Are the vectors viral, liposomal, and naked DNA vectors? Selected from the group consisting of:   In yet another embodiment, the ability to target molecules to dendritic cells And a vaccine comprising an antigen from a pathogen conjugated to a DEC-ligand Wherein the DEC-ligand is a carbohydrate and an anti-DEC antibody that binds DEC , As well as immunostimulants. Examples of pathogens are viruses, Examples include, but are not limited to, bacteria, parasites, and tumors. Immune sting The potent substance is selected from the group consisting of cytokines, lymphokines, and adjuvants. You may. In particular, the invention is poorly immunogenic or not at all immunogenic Efficient processing of molecules (DEC is an antigen processing mechanism of dendritic cells) As shown herein) and to reactive T lymphocytes Provides for targeting dendritic cells for presentation.   The invention also includes autoantigens or allergens conjugated to DEC-ligands. A composition that induces immunosuppression, wherein the DEC-ligand is a carbon that binds DEC. Selected from the group consisting of hydrates and anti-DEC antibodies, provided that the composition is an immunostimulant Subject to lack of quality. Stimulants, such as cytokines, lymphokines Dendritic cells with autoantigens or allergens without in or adjuvant By targeting them, quiescent dendritic cells process the antigen and provide Can be shown. Presentation of antigen by quiescent dendritic cells is an antigen-specific T cell annel And induces immune tolerance. Autoantigen is myelin basic Protein, collagen or its fragment, DNA, nucleoprotein, nucleolus Protein, mitochondrial protein, and pancreatic β-cell protein It may be selected from a group.   It is a main object of the present invention to provide ligands for DEC.   Therefore, an important direct object of the present invention is to identify ligands that specifically bind DEC. Is to determine.   Another object is to provide a nucleic acid encoding DEC.   Related objects include DEC, or the carbohydrate binding portion of the DEC lectin domain To express a nucleic acid encoding a part thereof.   These and other objects of the present invention are described in the following drawings, detailed description of the invention, And can be easily understood by referring to the examples.   BRIEF DESCRIPTION OF THE FIGURES FIG. The apparent mass of the antigen bound by NLDC-145 is 205 kDa. (A) Fixed NLDC-145 (right) (³⁵S) Methionine-cysteine labeled bone marrow DC extract Immunoprecipitation reveals an actively synthesized antigen with an apparent mass> 200 kDa . This antigen is not precipitated by immobilized non-specific rat IgG2a (left). (B) NLDC -145 binds a 205 kDa antigen in a non-reducing Western blot of crude thymus detergent extract. Combine. Three-fold serial dilutions of the extract starting at 0.17 thymus equivalents per lane at 5% (v / v) in the absence (left filter) or presence (right filter) of 2-mercaptoethanol The gel was loaded on a double gel and blotted onto nitrocellulose. filter Was probed with 10 μg / ml NLDC-145 IgG followed by peroxidase-conjugated anti-rat IgG . The staining pattern was visualized by ECL. No bands observed in reducing gel Was. STD: Pre-stained extensive traced from early filters to developed film Location of molecular weight marker (Bio-Rad) FIG. Summary: Purification of DEC-205 from the thymus All steps were performed at 0-4 ° C. Roy Peptin and PMSF were added to the ice-cold buffer just before use. FIG. Analysis of purified DEC-205 and process yield during purification (A) First Coomassie Stained with Brilliant Blue R-250 (left), then counterstained with silver (right) 8% acrylamide SDS-PAGE analysis of 5 μg of purified protein (B) 10 μg An equal volume of the major fraction from the purification stained with g / ml NLDC-145 IgG. S The fractions in the Tan Blot lanes 1-4 and 6-8 were transferred to the volume of the postnuclear supernatant. Dilution, so that the intensity of their 205 kDa band can be compared and the yield Could be estimated. Lane 10: small mA with apparent mass up to about 80 kDa b Systematic five-fold increase in antigen concentration to indicate a "ladder" of reactive bands : Nuc, nucleus; memb, membrane; extr, 0.5% NP-40 extract; nonads, column loading process Individual non-adsorbed fractions from early (nonads-5) and late (nonads-15) FIG. DEC-205 is an integral membrane protein with a pI of 7.5 (A) detergent, 1M K Cl or 100 mM Na_TwoCO_ThreeThymic membrane protein solubilized at pH 11.5 (lane 1) 2,3), and initially insoluble in high salt and high pH buffers, but then with detergent Immunoblot filter for proteins released from the membrane (lanes 4 and 5) Was stained with 10 μg / ml NLDC-145. (B) 10 μg of purified DEC-205 under denaturing conditions Single lane from a silver stained slab gel is shown with pH values adjacent to unstained Specified after elution of ampholytes from color lanes. FIG. Studies on carbohydrates bound to DEC-205 (A) DEC-205 is a glycoprotein . Purified 205 kDa protein, transferrin (Tf, positive control), and creatine Cre (negative control) was electroblotted onto nitrocellulose and the filters_Four To convert the vic-diol in the sugar to a fixed aldehyde. Digoxigenin (DIG) labeled hydrazide followed by anti-DIG conjugated to alkaline phosphatase Reaction with the antibody revealed the location of the glycoprotein on the blot. Transfer Similar to phosphorus, but unlike creatinase, DEC-205 stains for sugar I do. (B) The glycan of DEC-205 contains about 7 kDa of its apparent molecular weight. Apotrans Ferrin (aTf) and DEC-205 treated with trifluoromethanesulfonic anhydride (TMFSA) Non-selectively hydrolyze protein-bound carbohydrates (+) or untreated (-) did. Both of the processed proteins have apparent masses due to apotransferrin. Enhanced electrophoretic transfer corresponding to 5 kDa loss and 7 kDa loss by DEC-205 Mobility was shown. (C) FA of N-linked glycan released from DEC-205 by PNGaseF CE analysis 5.1 to 10.1 glucose units (Glc)_Five, (Glc)_Ten: Standard oligoglucose Eight bands that migrated between selected band positions in the jar were resolved. (D) D Exoglycosidase digestion of a mixture of N-linked glycans released from EC-205 And FACE analysis Lane 1: undigested N-linked oligosaccharide ((Glc) in lanes 1-5)_ThreeDarkness Lane 2: detergent artifact) Lane 2: digested with α-galactosidase   Lane 3: digested with α-galactosidase + NANaseIII Lane 4: previous 2 Lane 5: digestion with the three enzymes + β-galactosidase Lane 6 digested with N-acetylhexose aminidase: 2 times higher concentration of β Lane 7: before, except for -N-acetylhexose aminidase Lane 8: 2 times higher concentration of α- digested with the four enzymes + α-mannosidase Lane 9: Mannosidase except for mannosidase + α-fucosidase Silchitobiose core reference material: FC, fucosylated core; C, non-fucosylated core (E) Summary of findings from lectin staining and FACE analysis, including bisecting GlcNAc There are two fucosylated core structures, or without them. At the end Further heterogeneity results in the eight glycan variants observed in (C). FIG. N-terminal amino acid sequence of DEC-205 and Blotte with polyclonal antibody (A) amino-terminal sequence determined by two different core means (SEQ ID NO: 2) Synthesize a peptide spanning the first 19 residues and use it as an immunogen And KLH. (B) Prewash study: NLDC-145 is both polyclonal Specifically depletes the 205 kDa band detected by the antibody. Of crude thymic membrane extract Immunoblot Pass the extract twice through the NLDC-145 immunoaffinity column. Generated a depleted fraction and eluted material from the column. Filter 10 μg / ml NLDC-145 IgG, 0.1 μg / ml anti-N-terminal peptide IgG, and 0.1 μg / ml With anti-DEC-205 IgG. All three antibodies bind the same protein. FIG. Schematic diagram of DEC-205 FIG. Sequence of mouse DEC-205 and related proteins Predicted amino acid of mouse DEC-205 The acid sequence (SEQ ID NO: 3) was compared to the bovine PLA2 receptor sequence (SEQ ID NO: 4) and human Align with the sequence of the lophage mannose receptor (SEQ ID NO: 5). All three Amino acid positions that are identical in the species protein are shaded. Protein Dome Consensus to Isolate Ins and Form a C-Type CRD (Weis et al., Science 254: 1608-15) Amino acids are shown below the relevant sequence as follows: One-letter code for invariant amino acids, θ = aliphatic, χ = aliphatic or aromatic, φ = aromatic, Z = E or Q, B = D Or N, Ω = D, N, E or Q. Two hanging cysteines in CDR8 Is a^*Is emphasized. Automated Edman degradation from purified DEC-205 protein Defined peptide sequences are underlined and numbered (N indicates amino terminus , T indicates a peptide generated by trypsin, and L indicates endoproteinase lys-. C shows the peptide generated in C). FIG. Carboxyl-terminal cytoplasmic domain sequence of human DEC-205 (top) (SEQ ID NO: 1) Of the carboxyl-terminal cytoplasmic domain sequence of mouse DEC-205 (bottom) (SEQ ID NO: 6) Areas of comparison identity are underlined. Regions of similarity are shown in italics . FIG. DEC-205 expression DE in mouse tissues and transfected Cos-7 cells C. Expression of C-205 A. Northern blot symbol of poly-A + A extracted from indicated tissues : Br, brain mRNA; DC, dendritic cell mRNA: Ht, heart mRNA; Kd, kidney mRNA NA; Lv, liver mRNA; LN, lymph node mRNA; Sk, skin mRNA; Thm, breast Gland mRNA; tg, tongue mRNA FIG. DEC-205 endocytosis polyclonal rabbit anti-DEC-205 F (ab) ' 2 fragments and dendritic cells with 10 nm gold-labeled goat anti-rabbit IgG (Amersham) The DEC-205 ultrastructure analysis bar represents 100 μm. Symbol: MVV, multivesicular Lysosome; Lysosome; CP, coated pit; 0 ', fixed at time 0; ', Fixation after 1 minute incubation at 37 ° C; 5', ink for 5 minutes at 37 ° C Fixation after incubation; 20 ', fixation after incubation at 37 ° C for 20 minutes; 60 ′, Fixation after incubation at 37 ° C for 60 minutes FIG. Antigen presentation with rabbit anti-DEC-205 antibody or non-reactive rabbit antibody control Antigen presentation by incubated dendritic cells. Plotted against concentration of antibody in culture on several scales. Error bars are a measure of the mean. Indicates quasi-deviation. Symbol: Anti-DEC-205, rabbit anti-DEC-205 polyclonal IgG Cultures that received the determined amount; anti-IgG2a, IgG2a-specific polyclonal rabbit antibody Cultures that have received the indicated amount; cultures that have received the indicated amount of IgG, non-immune rabbit IgG Stuff FIG. Langerha with DEC-205 monoclonal and polyclonal antibodies Selective staining of sense cells Cultured epidermal cells are labeled with PE for class II MHC protein Doubly labeled with multiple antibodies of mAb (y-axis) and leukocyte antigen, followed by FITC anti-Ig (x-axis) Labeled. Average FITC fluorescence intensity for MHC-II (+) DC (eg, arrows in E and H) Is shown in the upper right corner of each panel. (A-D) Specificity: DEC, not for macrophage, B cell or T cell antigen -205 Langerhans cell staining Rat IgG2a hybridoma supernatant Was applied. (E-X) Drops of monoclonal and polyclonal reagents used in subsequent studies Set (E-G) Gradual dose of NLDC-145 mAb (0.6, 2, and 6 μg / ml) (H) Polyclonal non-immunized rat IgG2a (6 μg / ml) (I-X) rabbit IgG and F (ab ') at 3, 10, 30, and 100 μg / ml_TwoFragment Figure 14. Trypsin sensitivity and resynthesis of DEC-205 epitope Overnight culture of Langerhans cells (A-F) or lymph node B cells (G-L) for 30 minutes Exposure to 0.25% trypsin on ice or no treatment. lymph Node B cells had been stimulated with LPS to support survival. Cells immediately (d1) or Was further stained (d2) after one day of culture. Antibody is 30μg / ml anti-DEC-205 or non-immune F (ab ')_TwoFragments (A, C, E, G, I, and K); 2 μg / ml NLDC-145 or Or non-immunized rat IgG2 or anti-CD45 (clone M1 / 9, rat IgG2) hybrid Doma supernatant (B, D, F, H, J, and L). FIG. Fresh and cultured dendritic cells (arrows) from spleen and skin DEC-205 expression Spleen DC enriched in the low-density fraction of spleen cells was purified from anti-CD11c (y Axis, A-H), NLDC-145; anti-DEC-205 polyclonal F (ab ')_TwoFragment And counterstained with the corresponding non-immune control. Immediately after staining (fresh), or Performed after overnight culture. Class II MHC protein mAb identified in epidermal suspension Fresh and cultured Langerhans cells (y-axis, I-P) Shown for comparison. FIG. Class II MHC proteins by bone marrow DC grown from ancestors in the presence of GM-CSF Expression of protein and DEC-205 On days 6, 7, and 8 of culture, 10 μg / ml TIB- 120 (clone M5 / 114, anti-I-A^bdq, Anti-I-E^dk) Or non-immune IgG (A, C, E); 3 0 μg / ml anti-DEC-205 or non-immune IgG (B, D, F); or 2 μg / ml NLDC-14 5 (G, H, I), flow cytometry (A-F) and cell centrifugation Cells were examined by both ride (GI). (d6) On day 6, the cultures are unevenly leveled. Large growth factor that expresses class II MHC protein and little DEC-205 Vesicle aggregates. Remove aggregates from plastic adherent stromal cells Was. (D7, d8) Over two consecutive days of culture, aggregates were typical dendritic morphology, abundant Class II MHC proteins and numerous non-adherent cells with high levels of DEC-205 Vesicles were released. FIG. Expression of DEC-205 in peritoneal cells resident or indicated pro-inflammatory agent Peritoneal cells in the exudate induced by 30 μg / ml anti-DEC-205 or non-immune F (ab ')_Two Fragment and FITC anti-rabbit F (ab^')_TwoStained. The cells are then macrophaged Di, B and T cells were counterstained with PE labeled mAb. PE anti-Mac-1 / cd11b (mAb M1 / 7 07, y-axis) is shown here. Mac-1^brightCells are macrophages (arrow Ass) and Mac-1^dimCells are B cells (arrows), Mac-1 is negative cells, T cells It is. Abbreviations: ConA, concanavalin A; TGC, thioglycolate; BCG, Mycobacterium bovis Calmette Guerlain Figure 18. Expression of DEC-205 by leukocytes in fresh cell suspensions from three organs. Add an arrow to the cell. White cells from spleen (A-J), bone marrow (K-T), and peripheral blood (U-δ) A subset of blood cells was stained with PE-labeled antibody (y-axis) and non-immune (A-E, K-O , And U-Y) or anti-DEC-205 (F-J, P-T, and Z-δ) F (ab ')_TwoCounterstain with fragments Colored (x-axis, FITC). PE-labeled mAb is used for granule cells (RB6-8C5, anti-Gr-1), granule cells and Integrin CD11b (M1 / 70, anti-Mac-1), B cells (RA 3-6B2, anti-B220 / CD45RB), T cells (53-2.1, anti-Thy-1.2 / CD90), and class II MHC Protein (AMS-32.1, anti-I-A^d). FIG. Immunoblot bone marrow dendritic cells (BMDC), clot spleen leukocytes (SPL, about 65% B cells Cells) and resident peritoneal cells (PC, about 70% B cells, 30% macrophages) Gradual doses of cellular NP-40 extract were transferred to filters. Filter 10 μg / ml Stained with NLDC-145 IgG. BMDC has about 10 times more DEC-10 per cell than spleen B cells 5 and about 50 times more than peritoneal B cells. FIG. Immiscible mixing of DEC-205 antibodies with blocking dendritic cell stimulating activity in vitro Leukocyte reaction, in this case, gradual doses of mitomycin C treated spleen dendritic cells Continuous presence of 10 μg / ml of each of the indicated antibodies except 205 (this was used at 30 μg / ml) 3x10 below^ThreeAdded to allogeneic lymph node cells. Costimulatory protein B7-2 (clone GL-1 Only) mAb inhibited T cell proliferation. Anti-IgG: Surface Ig-related signaling of B cells Polyclonal negative control for protein (Sanchez et al., 1993, J. Exp. Med. 178: 1049 ) FIG. Expression of DEC-205 in thymus and lymph nodes (a-c): all at 10 μg / ml in monochrome Null NLDC-145 (a); polyclonal anti-DEC-205F (ab ')_TwoFragment (b); and polyc Thymus stained with lonal anti-DEC-205 IgG (c) and counterstained with hematoxylin Low output of cortex and medulla (M) Putative dendritic cells (arrowheads) are scattered in the medulla, The strongest thymic staining is in the cortical epithelium. (d-f): mAb NLDC-145 (d); polyclonal anti-DE C-205F (ab ')_TwoFragment (e); and polyclonal anti-DEC-205 IgG (f) In addition, mesenteric phosphorus showing B cell vesicles (B), T cell area of deep cortex (T), and medulla (M) Low power view of the parietal nodes. Darkly stained dendritic cells are distributed in the T cell area. (g-i): junction of thymic cortex and medulla (g), deep cortex of lymph node (h, venules Attached) and in B cell vesicles (i, no hematoxylin counterstaining) High power diagram to show distribution of 5 FIG. Expression of DEC-205 in spleen (a-c): B cell antibody (rabbit anti-IgG, a); DEC-2 05 antibody (polyclonal anti-DEC-205 IgG, b) and class II MHC protein Low power figure of splenic white medullary nodules stained with antibody (mAb M5 / 114, c) T cell area The central artery inside is marked with an arrow. T cell region contains few B cells (a, anti-IgG) However, a large number of scattered DEC-positive dendritic cells and class II MHC-positive dendritic cells (b-c) including. B cell vesicles are represented by "B" and marginal sinuses are represented by arrowheads. (d-e): mAb NLDC-145 (d); stained with polyclonal anti-DEC-205 (g), and anti-class II MHC (f) High power view of the splenic T cell region (the sheath around the artery and the central artery are marked with arrows) DEC-205 staining shows small spots in addition to more pronounced staining of dendritic cell bodies. Have quality. FIG. DEC-205 expression in some non-lymphoid organs (a-d): mAb NLDC-145 (a); Reclonal anti-DEC-205 F (ab ')_TwoFragment (b); and polyclonal anti-DEC-205 Brain capillaries (arrows, a-c) and small arteries (arrows, d) (e-h) stained with IgG (c-d): Lung, airway epithelium (arrows, e and h), isolated cells in lung parenchyma (arrowheads, g and h), And some putative alveolar macrophages (^*, H) shows anti-DEC-205 staining. class IIMHC protein (f) is not clear in airway epithelium, but many positive around airway There is a profile (Yajiri, f). (i): Bone marrow protruding lace stromal cells ( Arrow) expresses low levels of DEC-205. Dark staining of round cells is peroxidase Background staining with eosinophils expressing zeolite. (j): Tongue, example of stratified squamous epithelium A small number of putative Langerhans cells at the suprabasal level in the oral epithelium, shown as ) 1 shows DEC-205 staining. (k, l): Jejunum: DEC-205 is emitted by the resorbable epithelium of intestinal parenchyma. The highest level is manifested at the tip of the soft protrusion. Also, many cells in the basement membrane Stains darkly, but this staining is again a background of eosinophil peroxidase. It is. FIG. Tissue distribution of DEC-205 by immunoblotting Lysis of indicated organ Blot the material and filter with DEC-205 protein (A, mAb stained with NLDC-145) -) And LAMP-1 lysosomal membrane antigen (B, filter stained with mAb 1D4B) The current relative levels were compared. 50 μg of total protein was loaded in each lane. FIG. Amino acid and nucleotide sequence comparisons for human and mouse DEC-205 (A) Matrix of translated mouse (y-axis) and human (x-axis) amino acid sequences Plot (pam 250 matrix) The window plot for this plot is 6 0, the minimum% score is 60, and the hash value is 2. (B) Mouse (y-axis) And human (x-axis) DNA sequence matrix plot (DNA identity matrix). C) The window plot is 60, the minimum% score is 65, and the hash value Was 6 and the jump value was 1. Both strands were evaluated.   Detailed description of the invention   The invention is found primarily in dendritic cells, but also in B cells, brain capillaries, bone marrow strikes. Roma, intestinal parenchyma and lung airway epithelium, and thymic cortical epithelium and peripheral lymphoid organs Endogenous, referred to herein as "DEC", found in dendritic cells in the T cell region of Regarding membrane proteins. In addition, trace amounts of this protein are found in the liver, heart, and kidney. It is found in organs such as the gut. Mouse and human counterparts of DEC are 205 kDa Has an apparent molecular weight of 7.5, the mouse counterpart has an isoelectric point at pH 7.5, and the carbohydrate is Contains about 7 kDa of the total mass of DEC. There are eight different carbohydrates, but two related It is clear that it consists of tena N-linked glycans and no O-linked glycans are present. No. The protein is found mainly in dendritic cells and thymic epithelial cells and has a 205 kDa Because of its molecular weight it is called DEC-205. Mouse counterpart of DEC and human Characterization of gut counterparts indicates the presence of 10 carbohydrate binding domains with a high degree of homology , DECs from other species have more or fewer such domains. It is possible to get. Similarly, DEC can be used in other cell types, such as other cell types. It can be expressed by epithelial cells from a tissue or organ.   In addition, the present invention provides for the cloning and encoding of the gene encoding DEC-205. Protein characterization. Sequence information shows that DEC-205 has 10 C-type lectins. Indicates that it is a receptor with a main, which is a macrophage mannose Receptors and other related agents that bind carbohydrates and mediate endocytosis Homologous or similar to the receptor. The human counterpart has a lectin domain. It is clear to have. Therefore, DEC has a corresponding number of lectin domains. And may be involved in antigen processing by dendritic cells.   Still another aspect of the present invention relates to a human DEC protein and a gene encoding the same. Identification.   DEC based on deduced amino acid sequence information obtained from cloning of dec cDNA Another feature of the protein is its unique coating pit localization core in the cytoplasmic tail of the protein. A consensus domain or motif, which, as shown in FIG. There are regions of homology and regions of difference between the two counterpart proteins.   In addition, DEC-205 is rapidly internalized by coated vesicles and binds Multivesicular endosomal compartment resembling vesicles containing MHC class II involved in processing Was found to be sent to the picture. Further, the present invention provides a rabbit antibody specific to DEC-205. The body is efficiently processed by dendritic cells and submitted to rabbit-specific T cell clones. It is based, in part, on further findings that have been shown.   Therefore, and most importantly, the present invention relates to additional ligands for the DEC-205 receptor. The identification of dendritic cells and other cells with DEC-205. Vesicles can be advantageously targeted. Antigen targets for presentation by dendritic cells Setting is tolerant when dendritic cells are at rest, or if dendritic cells are For example, cytokines or lymphokines, such as colony stimulating factor (CSF) Can provide an immune stimulus (ie, vaccination) when activated by a stimulus You.   Furthermore, the presence of DEC in epithelial cells, for example, is extracellular in lung respiratory epithelium, respectively. From the lateral surface to the lung airways or from the basolateral surface of the intestinal epithelium to the intestinal lumen, Transepithelial transfer of molecules from the apical (luminal) surface of their epithelium to the pulmonary or intestinal circulation Suggest an important role in transport. Thus, the present invention provides a pharmacological For pulmonary or intestinal infections by targeting biological agents to DEC Parenteral delivery of physical agents, such as antibiotics, and aerosol application and pulmonary Provides systemic delivery of pharmacological agents by inhalation or intestinal uptake and absorption .   In yet another aspect, the present invention provides a blood-brain barrier with DECs located in brain capillaries. Provides pharmacological drug targeting across the phylum.   Therefore, various terms having the following meanings are used herein.   The term “candidate ligand” is a test for the ability to specifically bind to DEC Used herein to represent the molecule under consideration. Is explained in great detail below As such, candidate ligands include saccharides (ie, sugars, carbohydrates, or glycans). But not limited thereto. Also, as used herein, " The term "ligand" may refer to an antibody reactive with DEC.   A molecule is an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or Is "antigenic" when it can specifically interact with T cell antigen receptors is there. The antigenic polypeptide has at least about 5, preferably at least about 10, amino acids. Nonoic acid. The antigenic portion of the molecule is immune for antibody or T cell receptor recognition. It may be that part that is epidemiologically dominant, or that it immunizes the antigenic part Used to produce antibodies for that molecule by conjugation to the carrier molecule. Part. A molecule that is antigenic need not itself be immunogenic. That is, it need not be possible to elicit an immune response without a carrier.   A composition comprising "A" (where "A" is a single protein, DNA molecule, vector) , Recombinant host cells, etc.), proteins, DNA, vectors in the composition. At least about 75% by weight (depending on the category of species to which A and B belong) When "A", "B" is not substantially contained (in this case, "B" is one or more types of dirt). Dyed proteins, DNA molecules, vectors, etc.). “A” is A + in the composition Make up at least about 90% by weight, most preferably at least about 99% by weight of species B Is preferred. In addition, compositions that are substantially free of contaminants are active in the species involved. Or it is preferred to include only a single molecular weight species with features.   The term “pharmaceutically acceptable” refers to a substance that is physiologically tolerated and administered to a human. Sometimes allergic or similar adverse reactions, such as stomach upset, dizziness, etc. Represents molecular entities and compositions that do not naturally occur. In particular embodiments, herein The term “pharmaceutically acceptable” as used shall refer to any federal or state More approved or USP for use in animals, more particularly humans Or other listed Pharmacopoeia. “Bear The term "body" refers to a diluent, adjuvant, excipient, or Represents a vehicle. Such pharmaceutical carriers may be sterile liquids, such as water and oils (petroleum, Crude oil, vegetable oil or synthetic source oil such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Included). Water or aqueous solution, saline solution and aqueous dextrose solution Liquid and glycerol solutions may be used as carriers, particularly for injectable solutions. preferable. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W.Martin. ing.   The term “therapeutically effective amount” refers to a clinically significant lack of host activity, function, and response At least about 15%, preferably at least 50%, more preferably at least 90% %, As used herein to mean an amount sufficient to reduce, most preferably inhibit, Is done. A therapeutically effective amount is also sufficient to produce a clinically significant improvement in the host's symptoms. is there.   The term “adjuvant” refers to a compound that enhances the immune response to an antigen or Represents a mixture. Adjuvants are used as tissue depots that gradually release the antigen, Can be used as a lymphoid activator to nonspecifically enhance the immune response (Hood Et al., Imunology, Vol. 2, 1984, Benjamin / Cummings: Menlo Park. California , 384). Often, primary antigen administration with antigen alone is performed in the absence of an adjuvant tongue. Will not be able to elicit humoral and cellular immune responses. A Complete Freund's adjuvant, incomplete Freund's adjuvant , Saponins, inorganic gels, such as aluminum hydroxide, surface active substances, such as , Lysolecithin, pluronic polyol, polyanion, peptide, oil or charcoal Hydrogen emulsion, keyhole limpet hemocyanin, dinitrophenol And potentially beneficial human adjuvants, such as BCG (Calmet Mycobacterium tuberculosis) and Corynebacterium parvum But not limited to these. Adjuvants are preferably pharmaceutically acceptable No.   Various abbreviations used herein include PBS, phosphate buffered saline; mAbs, Monoclonal antibody; SPF, free of certain pathogens; PMSF, phenylmethyls Ruphonyl fluoride; DIFP, diisopropyl fluorophosphonate; FACE, fluorescence Body assisted carbohydrate electrophoresis.   Encodes DEC, or a fragment, derivative, chimera or analog thereof Gene to do   The present invention relates to any animal, especially mammals or birds, and more particularly from human origin. Functional part of the DEC receptor of the invention (full length of DEC, or naturally occurring form) And the isolation of the gene encoding the antigenic fragment thereof. You. The term "gene" as used herein encodes a polypeptide Represents a collection of nucleotides and includes cDNA and genomic DNA nucleic acids. Special below In certain embodiments, the specific nucleotide sequence of human DEC encoding DNA (SEQ ID NO: 8) and the amino acids shown in SEQ ID NO: 3 and SEQ ID NO: 8, respectively. Deduced coding sequences for both mouse and human DEC polypeptides having a noic acid sequence Is provided.   Therefore, conventional molecular biology, microbiology, and recombinant DN within the state of the art. A technology may be used. Such techniques are explained fully in the literature. An example For example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manu al, Part 2 (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor , New York (herein "Sambrook et al., 1989"); DNA Cloning: A Practical A pproach, Volumes 1 and 2 (edited by D.N. Glover, 1985); Oligonucleotide Synthesis ( M.J.Gait Editing, 1984); Nucleic Acid Hybridization [B.D.Hames & S.J.Higgin s Edit (1985)]; Transcription And Translation [B.D.Hames & S.J.Higgins Editing (1984)]; Animal Cell Culture [R.I.Freshney Editing (1986)]; Immobilized  Cells And Enzymes [IRL Press (1986)]; B. Perbal, A Practical Guide To Mo lecular Cloning (1984); edited by F.M.Ausubel et al., Current Protocols in Molecula r Biology, John Wiley & Sons, Inc. (1994).   Therefore, as appearing herein, the following terms should have the following definitions .   A “nucleic acid molecule” is a single-stranded or double-stranded helix of a ribonucleoside (ad Nosin, guanosine, uridine or cytidine; "RNA molecule") or deoxy Ciribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymine Gin or deoxycytidine; "DNA molecule") phosphate polymer form Express state. Double-stranded DNA-DNA, DNA-RNA and RNA-RNA helices Is possible. The term nucleic acid molecule, especially a DNA molecule or an RNA molecule, Represents only the primary and secondary structure of the molecule and limits it to any particular tertiary form Not determined. Thus, the term refers, inter alia, to linear or circular DNA molecules ( Double-stranded DNA found in, for example, restriction fragments), plasmids, and chromosomes including. In describing the structure of particular double-stranded DNA molecules, the sequence 5 ′ along the strand (ie, a strand having a sequence homologous to the mRNA) It may be described herein according to the usual convention of showing only the sequence in the 3 'direction. "Recombinant A "DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation.   The single-stranded form of the nucleic acid molecule can be converted to other cores under appropriate conditions of temperature and solution ionic concentration If the nucleic acid molecule can anneal to an acid molecule, then the nucleic acid molecule is another nucleic acid molecule, "Can hybridize" to nome DNA or RNA (Sambroo See k et al., supra). Conditions for temperature and solution ion concentration are hybridization Determine the “stringency” of the solution. Preliminary script for homologous nucleic acids Low stringency hybridization corresponding to a Tm of 55 ° Solution conditions can be used (eg, 5x SSC, 0.1% SDS, 0.25% milk, And no formamide; or 30% formamide, 5x SSC, 0.5% SDS). Suitable High stringency hybridization conditions are higher Tm, for example, 4 Equivalent to 0% formamide, 5x or 6x SSC. High stringency hive Redidation conditions are the highest Tm, eg, 50% formamide, 5x or 6x  Equivalent to SSC. Hybridization means that two nucleic acids contain complementary sequences But depending on the stringency of the hybridization, A mismatch between the two is possible. Stringers suitable for hybridizing nucleic acids The frequency depends on the length of the nucleic acids and the degree of complementarity, variables known in the art. 2 types The greater the degree of similarity or homology between nucleotide sequences of The value of Tm for a hybrid of nucleic acids having Nucleic acid hybridization Relative stability (corresponding to high Tm) decreases in the following order: RNA: RNA, DNA: RNA, DNA: DNA. Greater than 100 nucleotides in length For hybrids, equations for calculating Tm have been derived (Sambrook et al., Supra). Bibliography, 9.50-0.51). Uses short nucleic acids, ie oligonucleotides For hybridizations where mismatches are important, The length of the leotide determines its specificity (see Sambrook et al., Supra, 11.7-11.8. See). The minimum length of nucleic acids that can form a hybrid is small Both are preferably 10 nucleotides. At least about 15 nucleotides Is more preferred. Most preferably it is at least about 20 nucleotides in length preferable.   DNA "coding sequences" are used in vitro when placed under the control of appropriate regulatory sequences. Or a double-stranded DNA sequence that is transcribed in cells in vivo and translated into a polypeptide It is. The boundaries of the coding sequence are defined by the initiation codon at the 5 '(amino) terminus and the 3' (carboxy) G) determined by the translation stop codon at the end. Prokaryotic sequence, true as coding sequence CDNA from eukaryotic mRNA, gene from eukaryotic (eg, mammalian) DNA Nome DNA sequences, and even synthetic DNA sequences, including but not limited to Absent. If the coding sequence is intended for expression in eukaryotic cells, a polyadenylation system may be used. Signal and transcription termination sequences will usually be located 3 ′ to the coding sequence.   Expression control sequences, eg, transcription control sequences and translation control sequences, are DNA control sequences, eg, For example, promoters, enhancers, terminators, etc. Provides expression of the coding sequence in the cell. In eukaryotic cells, polyadenylation signal Is a regulatory sequence.   A “promoter sequence” is capable of binding RNA polymerase in a cell. , A DNA regulatory region capable of initiating transcription of a downstream (3 'direction) coding sequence . For the purpose of specifying the present invention, the promoter sequence has a 3 ′ And extends upstream (5 'direction) to detectable levels above background Contains the minimum number of bases or elements required to initiate transcription at Promoter arrangement Within the sequence, the transcription start site (eg, by mapping with nuclease S1) Protein binding domain responsible for binding RNA polymerase) The main (consensus sequence) will be seen.   The coding sequence is used when RNA polymerase transcribes the coding sequence into mRNA. In the cell "under control" of the transcriptional and translational regulatory sequences, or Which is subsequently trans-RNA spliced into the coding sequence. Translated into the protein encoded by the sequence.   "Signal sequence" is the beginning of the coding sequence for the protein to be expressed on the cell surface include. This sequence encodes the N-terminal signal peptide for the mature polypeptide It induces host cells to translocate the polypeptide. “Tran The term “slocation signal sequence” refers to this type of signal sequence in the book. Used in the specification. Translocation signal sequences are eukaryotic and prokaryotic Are found in association with various native proteins and are often found in both types of organisms. Ability.   As used herein, the term “sequence homology” in all grammatical forms refers to Tan from the superfamily (eg, the immunoglobulin superfamily) Including homologous proteins from protein and heterologous (eg, myosin light chain, etc.) Represents relationships between proteins with a “common evolutionary origin” (Reeck et al., 1987, Cell5 0: 667).   Therefore, the term “sequence similarity” in all grammatical forms is a common evolutionary origin Identity or identity between nucleic acid or amino acid sequences of proteins that do not share the same (See ReecK et al., Supra).   The two DNA sequences have at least about 75% (preferably less) of that nucleotide. At least about 80%, most preferably at least about 90 or 95%) "Substantially homologous" or "substantially similar" if they fit over a given length It is. Substantially homologous sequences can be obtained using conventional software available in the Sequence Data Bank. Using software, or, for example, a stream identified for that particular system. In Southern hybridization experiments under transient conditions, these sequences Can be identified by comparing Identification of appropriate hybridization conditions Within the technical level of the industry. See, for example, Maniatis et al., Supra; DNA Cloning, I. See Volumes and II, supra; Nucleic Acid Hybridization, supra.   Similarly, two amino acid sequences have more than 70% identical or functional amino acids "Substantially homologous" or "substantially similar" when they are identical. As well Is a homologous sequence, for example, GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) Identified by the alignment used.   The term "corresponding" is used herein to describe similar or homologous sequences The exact position is the same or different from the molecule whose similarity or homology is to be measured. I don't care. Thus, the term "corresponds" indicates sequence similarity and It does not indicate the numbering of the acid residues or nucleotide bases.   The gene encoding DEC (whether genomic DNA or cDNA) is It can be isolated from any source, especially human cDNA or genomic libraries. DEC Methods for obtaining genes are known in the art, as described above (e.g., Sambrook et al.). 1989, supra). In a special embodiment described below, the mouse DEC-2 The cDNA encoding 05 is isolated from a dendritic cell library. In addition, Mau Dec cDNA and mouse genomic dec cDNA Used to isolate genes.   Therefore, animal cells are a potential source of nucleic acids for molecular cloning of the dec gene. Can be used locally. The DNA is cloned DNA (for example, DNA “Library”). Lee ") may be obtained by conventional procedures known in the art, preferably Is a genomic DNA purified by chemical synthesis, cDNA cloning, or purified from dendritic cells. High level of protein by cloning NA or its fragment CDNA library prepared from a tissue having the expression of cDNA or thymic epithelial cDNA libraries, these are DEC Because it is the cell that demonstrates the highest level of expression of Sambrook et al., 1989, supra; Glover, D.M. Editing, 1985, DNA Cloning: A Prac- tical Approach, MRL Press, Ltd., Oxford, U.K.I, II). Get A clone derived from nome DNA has a regulatory region and an intron DN in addition to a coding region. A region may be included. Clones derived from cDNA contain intron sequences. Will not. Regardless of the origin, the gene is suitable for gene transfer. Should be molecularly cloned into the vector.   In molecular cloning of genes from genomic DNA, DNA fragments Will be generated, some of which will encode the desired gene. DNA is various It may be cleaved at a specific site using a restriction enzyme. Also in the presence of manganese DNAse may be used to fragment the DNA, or the DNA may be sonicated, for example. It can be physically sheared by the treatment. The linear DNA fragment is then And polyacrylamide gel electrophoresis and column chromatography Can be separated according to size by conventional techniques, but not limited thereto.   Once the DNA fragment is generated, a specific DN containing the desired dec gene Identification of the A fragment can be performed in several ways. For example, a certain amount of the dec gene Or a specific RNA thereof, or a fragment thereof is available and purified If the DNA fragment can be labeled, Can be screened by hybridization (Benton and Davis, 1977, Science 196: 180; Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A.7. 2: 3961). For example, in the partial amino acid sequence information obtained for the DEC protein, A corresponding set of oligonucleotides could be prepared and were performed in the specific examples below. As a probe for DNA encoding DEC, or cDNA or mR NA primers (eg, in combination with RT-PCR poly-T primers) Can be used. Fragments that are highly specific for the DEC of the present invention may be selected. preferable. Those DNA fragments which have substantial homology to the probe Will form a hybrid. As mentioned above, the greater the degree of homology, More stringent hybridization conditions may be used. Special implementation In one embodiment, the human dec cDNA is derived from the 3 'coding sequence of the mouse dec cDNA. It was cloned using a derived 300 base pair probe. Human cDNA is high B using stringency hybridization conditions (0.1 SSC, 65 ° C) Obtained from a lymphoma library. Thus, high stringency hybridization Advantages of the solicitation conditions identify homologous dec genes from other species You.   For example, a protein product wherein the gene has an isoelectric focusing electrophoretic amino acid composition Or encoding a partial amino acid sequence of a DEC protein disclosed herein. If so, further selections can be made based on the nature of the gene. Thus, the presence of the gene Assays based on the physical, chemical, or immunological properties of the expression product Can be detected. For example, cDNA clones or appropriate mRNA DNA clones for lid selection can be selected, these are, for example, Similar or identical electrophoretic transfer, isoelectric focusing or non-planar as known Equilibrium pH gel electrophoresis behavior, proteolytic digestion map, or antigenic tan Produces protein. For example, rabbit polysaccharide of mouse DEC described in detail below. Clonal antibodies confirm expression of DEC, both mouse and human counterparts Can be used to In another aspect, it has an apparent molecular weight of 205 kDa and is specifically Tamper that is digested to produce a specific ladder (not smear) of low molecular weight bands Quality is a good candidate for DEC.   In addition, the dec gene of the present invention can be used for mRNA selection, that is, nucleic acid hybridization. Alternatively, it can be identified by translation in vitro. In this operation, the nucleotchi Fragment is used to isolate complementary mRNA by hybridization. Used. Such DNA fragments correspond to the available purified dec DNA. Or synthetic oligonucleotides designed from partial amino acid sequence information It may be. Immunoprecipitation analysis of in vitro translation products of isolated mRNA products Or a functional assay (eg, tyrosine phosphatase activity) Then, a complementary DNA fragment containing the desired sequence is identified. In addition, specific mR NA is an immobilized antibody specifically directed against DEC, e.g., as described herein. Polyisolated from Cells to a Rabbit Polyclonal Anti-Mouse DEC Antibody Can be selected by adsorption of the system.   The radiolabeled dec cDNA was used as the template for the selected mRNA (adsorbed polysodium). From the homepage). Then radiolabeled mRNA or cDNA Homologous dec DNA fragment among other genomic DNA fragments Can be used as a probe to determine   Further, the present invention relates to the present invention, which has the same or homologous functional activity as DEC. Genes encoding DEC analogs and derivatives, and their congeners from other species A cloning vector containing the gene. Derivatives and analogs for DEC Production and use are within the scope of the present invention. In a specific embodiment, the derivative or An analog is functionally active, that is, one or more of the features associated with full-length wild-type DEC. Active activity.   In another aspect, the DEC protein of the invention is found in DEC-205 A lectin domain or another protein, e.g., macrophage mannose Substituting a domain from the receptor or muscle phospholipase receptor Can be prepared.   DEC derivatives are modified by substitutions, additions or deletions to give functionally equivalent molecules. By changing the nucleic acid sequence to be applied. Promote or increase natural DEC It is preferred that derivatives with increased functional activity be produced. Also this Such derivatives are the same or larger for the natural ligands of the DECs of the invention. Encoding a soluble fragment of the DEC extracellular domain with affinity You. Such soluble derivatives are potential inhibitors of the ligand binding to DEC -Can be.   Due to the degeneracy of the nucleotide coding sequence, substantially the same Other DNA sequences encoding amino acid sequences may be used in the practice of the present invention. this Alleles, homologous genes from other species, and the same amino acid in the sequence Altered by substitution of a different codon encoding an acid residue, thus silent alteration Nucleotide sequence containing all or part of the dec gene However, the present invention is not limited to these. Similarly, as the DEC derivative of the present invention, a primary amino acid As a sequence, functionally equivalent amino acid residues are replaced by residues in the sequence and conserved amino acids All of the amino acid sequence of the DEC protein including the altered sequence resulting in an acid substitution Ma Or a part thereof, but is not limited thereto. For example, in the array One or more amino acid residues can be replaced by another amino acid of similar polarity, Acts as a noh equivalent, producing a silent change. Of amino acids in the sequence Substitutions may be selected from other members of the class to which the amino acid belongs. For example , Non-polar (hydrophobic) amino acids such as alanine, leucine, isoleucine, bali , Proline, phenylalanine, tryptophan and methionine . Glycine, serine, threonine, cysteine, tyro as polar neutral amino acids Syn, asparagine, and glutamine. Positively charged (basic) Amino acids include arginine, lysine and histidine. Negatively charged (Acidic) amino acids include aspartic acid and glutamic acid. Such changes are measured by polyacrylamide gel electrophoresis or isoelectric point. Would not be expected to affect the apparent molecular weight.   Genes encoding the DEC derivatives and analogs of the present invention are known in the art. It can be produced by various methods. The operations that lead to their production are at the genetic level or Can occur at the protein level. For example, cloned gene sequences are It can be modified by any of a number of known strategies (Sambrook et al., 1989, supra). Literature). The sequence can be cleaved at the appropriate site with one or more restriction endonucleases. Followed by further enzymatic modification if desired, isolated and tethered in vitro . In the production of a gene encoding a derivative or analog of DEC, a modified gene Is interrupted by a translation termination signal in the gene region encoding the desired activity. To ensure that they remain in the same translational reading frame as the DEC gene Attention should be paid.   In addition, the nucleic acid sequence encoding DEC may be mutated in vitro or in vivo. To generate and / or degrade translation, initiation, and / or termination sequences, or Changes in the coding region and / or forms new restriction endonuclease sites. Formed or degrade existing ones to further facilitate in vitro modification. like this Such mutations preferably enhance the functional activity of the mutated DEC gene product. New Also, fragments of DEC, such as one or a few lectin domains Deletion mutants encoding amino acids can be generated (Taylor et al., 1992, J. Biol. Chem. 267: 1719). In vitro site-induced mutagenesis (Hutchinson, C. et al., 1978, J. Blol. Chem. 253: 6551; Zoller and Smith, 1984, DNA 3: 479-488; Oliphant et al. Hutchinson et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83: 7 Ten), Any technique of mutagenesis known in the art can be used. PCR technology for site induction Preferred for guided mutagenesis (Higuchi, 1989, “Using PCR to Engineer DNA”, PCR Technology: Principles and Applications for DNA Amplification, H.Erl ich, edited by Stockton Press, Chapter 6, pages 61-70).   The identified and isolated gene is then inserted into a suitable cloning vector. obtain. Many vector-host systems known in the art can be used. Possible vector Include, but are not limited to, plasmids or modified viruses. And the vector system must be compatible with the host cell used. vector Examples of E. coli, bacteriophages such as λ derivatives, or plasmids For example, pBR32 derivative or pUC plasmid derivative, for example, pGEX vector , Pmal-c, pFLAG, and the like, but are not limited thereto. Cloning For insertion into a vector, for example, a DNA fragment is inserted into a cloning vector (this Which have complementary cohesive ends). However, D Complementary restriction sites used to fragment NA are present in cloning vectors Otherwise, the ends of the DNA molecule may be enzymatically modified. Also, any desired The site is created by connecting a nucleotide sequence (linker) to the end of the DNA. It may be. These tethered linkers contain restriction endonuclease recognition sequences. It may include a specially synthesized oligonucleotide that encodes. Recombination Molecule is transformation, transfection, infection, electroporation, etc. Can be introduced into host cells, resulting in many copies of the gene sequence . The cloned gene is preferably contained in a shuttle vector plasmid, This can be done in cloning cells, for example in E. coli, and transfer to a suitable expression cell line. To provide easy purification (if such is desired) for subsequent insertion. For example, a shuttle vector (which can replicate in more than one type of organism) The vector from the E. coli plasmid is a yeast 2μ plasmid. E. coli and Saccharomyces cerevisiae (Sacc- haromyces cerevisiae).   Alternatively, the desired gene is a suitable gene in a "shotgun" approach. It may be identified and isolated after insertion into a cloning vector. For example, rhino Enrichment of the desired gene by fractionation can be performed before insertion into the cloning vector. You.   Expression of DEC polypeptide   DEC, or an antigenic fragment, derivative or analog thereof, or Suitable nucleotide sequences encoding functionally active derivatives, including chimeric proteins Transcription and translation of an expression vector, ie, a sequence encoding an inserted protein Can be inserted into a vector containing the necessary elements. Such elements are referred to herein as “Pro Thus, the nucleic acid encoding the DEC of the present invention is referred to as “motor”. It is operatively associated with a promoter in an expression vector. cDNA sequence and Geno Both gene sequences have been cloned and can be expressed under the control of such regulatory sequences. Ma In addition, the expression vector preferably contains an origin of replication.   Necessary transcription and translation signals can be provided in a recombinant expression vector, and Are supplied by native genes encoding DEC and / or its flanking regions May be done.   As potential host-vector systems, viruses (eg, vaccinia virus, A mammalian cell line infected with an adenovirus or the like; a virus (eg, baculo Ills); an insect cell line; a microorganism such as yeast containing a yeast vector; Is bacteriophage, DNA, plasmid DNA, or cosmid DNA Include, but are not limited to, transformed bacteria. Vector Expression elements vary in their strength and specificities. Host-vector used Depending on the system, any one of several suitable transcription and translation elements may be used I can do it.   A recombinant DEC protein of the present invention, or a functional fragment or derivative thereof, The chimeric construct, or analog, is chromosomal after integration of the coding sequence by recombination. Can be expressed. In this regard, one of several amplification systems has a high level of stability (See SambrooK et al., 1989, supra). thing).   Cells into which the recombinant vector containing the nucleic acid encoding DEC has been introduced The cells are cultured in a suitable cell culture medium under conditions that give the expression of DEC.   Already described for insertion of DNA fragments into cloning vectors Any of the methods encodes appropriate transcription / translation control signals and proteins It can be used to construct an expression vector containing a gene consisting of a sequence. These people Methods include in vitro recombinant DNA and synthetic techniques and in vivo recombination (gene Replacement).   Expression of the DEC protein is determined by any promoter / enzyme known in the art. These regulatory elements may be regulated by a Hanser element, but in the host chosen for expression. Must be functional. Promoters that can be used to regulate DEC gene expression SV40 early promoter region (Benoist and Chambon, 1981, Nature 29) 0: 304-310), a promoter contained in the 3 'long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22: 787-797), herpes thymidine kinase promoter -(Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78: 1441-1445), metallothiose Regulatory sequences of the nein gene (Brinster et al., 1982, Nature 296: 39-42), prokaryotic Current vectors, such as the β-lactamase promoter (Villa-Kamaroff et al., 1978) Proc. Natl. Acad. Sci. U.S.A. 75: 3727-3731), or the tac promoter (DeBoe r et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 21-25): “Useful proteins fr omrecombinant bacteria ", Scientific American, 1980, 242: 74-94. And promoter elements from yeast or other fungi, such as the Gal4 promoter. Promoter, ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycero) Kinase) promoter, alkaline phosphatase promoter, and Transcriptional regulatory regions (these show tissue specificity and are used in transgenic animals) But not limited thereto.   The expression vector containing the nucleic acid encoding the DEC of the present invention can be prepared by four general protocols. Approaches: (a) PCR amplification of desired plasmid DNA or specific mRNA, (b) nucleic acid Hybridization, (c) the presence or absence of selectable marker gene function, and ( d) can be identified by expression of the inserted sequence. In the first approach, the nucleic acid is It can be amplified by PCR to provide detection of the amplification product. In the second approach, The presence of the foreign gene inserted into the expression vector correlates with the inserted marker gene. Detection by nucleic acid hybridization using a probe containing the same sequence I can do it. In a third approach, the recombinant vector / host system comprises Certain “selectable marker” gene functions (eg, β- Galactosidase activity, thymidine kinase activity, antibiotic resistance, transformation Phenotype, occlusal formation in baculovirus, etc.) Defined and can be selected. In another example, the nucleic acid encoding DEC is a "selection" vector. When inserted within the marker "gene sequence, the recombinant containing the DEC insert is It can be identified by the absence of DEC gene function. In a fourth approach, recombinant Expression vectors assume a conformation in which the expressed protein is functionally active Provided that the activity and biochemical characteristics of the recombinantly expressed gene product Or by analyzing for immunological characteristics.   Once a particular recombinant DNA molecule has been identified and isolated, it is known in the art. Several methods may be used to propagate it. Suitable host systems and growth conditions Once established, the recombinant expression vector can be propagated and prepared in predetermined quantities. Destination As described in the above, the following vectors or These derivatives include, but are not limited to. A few people are human Or an animal virus, such as a vaccinia virus or an adenovirus, an insect virus. Ils, eg baculovirus, yeast vector, bacteriophage vector (Eg, λ), and plasmid and cosmid DNA vectors.   In addition, it modulates the expression of the inserted sequence or produces the gene in a particular desired fashion. The host cell strain to be modified and processed can be selected. Different host cells Processing and modification of protein translation and post-translation (eg, glycosylation, [eg For example, it has a characteristic and specific mechanism for the [cleavage] of the signal sequence. Appropriate cell lines or host systems are used to modify and modify the expressed foreign protein as desired. Selected to ensure sexing. For example, expression in bacterial systems It can be used to produce a cosylated core protein product. However, Bakte Transmembrane DEC protein expressed in the rear You may not. Expression in yeast can result in glycosylation products, but glycosylation The turns will probably be different from those obtained by expression in mammalian cells. Expression in eukaryotic cells is responsible for "natural" glycosylation and folding of heterologous proteins Possibility may be increased. Additionally, mammalian cells, such as Chinese hamster ovaries (CHO), Alrican Green Monkey COS cells and fibroblast NIK-3T3 cells Expression in cells (eg, 293 cells) to regenerate or constitute DEC activity Means can be provided. In addition, different vector / host expression systems are Reactions can be affected to different extents, for example, proteolytic cleavage.   In a preferred aspect of the invention, the DEC is a DEC-targeted resource. Model epithelial cells for studying the efficacy and rate of transepithelial migration of gand or molecule, For example, it is introduced into Mazin-Darby canine kidney (MDCK) cells. Also shown below Dec gene is transformed by in vivo or ex vivo gene transfer. It can be introduced into epithelial cells or dendritic cells for treatment.   Vectors can be constructed using methods known in the art, such as transfection, Tropoporation, microinjection, transduction, cell lysis Dextran, calcium phosphate precipitation, lipofection (lysosomal Fusion), use of a gene gun, or a DNA vector protein transporter (eg, Wu Et al., 1992, J. Biol. Chem. 267: 963-967; Wu and Wu, 1988, J. Biol. Chem. 263: 146 21-14624; Canadian Patent Application No. 2,012,311 filed on March 15, 1990 by Hartmut et al.  ) Into the desired host cell.   Recombinant DEC protein expressed as integral membrane protein Can be more isolated and purified. Generally, integral membrane proteins clean their membranes with detergents, e.g. For example, sodium dodecyl sulfate (SDS), Triton X-100, Nonidet P-40 (NP-40 ), Digoxin, sodium deoxyfolate, etc. (including their mixtures) But not limited thereto). Solubilization is the sonication of suspensions May be more advanced. In a specific embodiment described below, DEC-205 contains 0.5% NP-40. It is solubilized from the thymic membrane pellet in the containing buffer. The soluble form of the protein is Collect the culture broth or, for example, treat with detergent, and optionally sonicate as described above. It is obtained by solubilizing the inclusion bodies by processing or other mechanical methods. Solubilized or soluble proteins can be obtained by various techniques, for example, poly Acrylamide gel electrophoresis (PAGE), isoelectric focusing, 2-dimensional gel electrophoresis, Chromatography (eg, ion exchange chromatography, affinity Chromatography, immunoaffinity chromatography, and cydin Column chromatography), centrifugation, differential solubility, immunoprecipitation, Or can be isolated by any other conventional technique for protein purification . In a specific embodiment described below, DEC-205 is a monoclonal antibody that binds NLDC-145. Purified using an affinity column containing In another embodiment below DEC-205 was purified by immunoprecipitation with monoclonal antibody NLDC-145.   Characterization of DEC structure   Once a recombinant expressing the DEC gene sequence has been identified, Once present, the recombinant DEC product can be analyzed. This is the radioactive labeling of the product, Followed by physical properties of the product, including analysis by gel electrophoresis, immunoassay, etc. And functionality based assays.   For example, endocytosis and coating pits, and thus Class II MHC processing To endocytic vesicles by endocytosis associated with phasing Contains the expressed protein or its cytoplasmic domain that mediates The ability of the fragment can be measured. In one embodiment below, an endosite The cis was analyzed using an anti-DEC antibody and a gold-labeled secondary antibody reactive with the anti-DEC antibody. Evaluated by microscope. In another embodiment, processing and presenting the antigen That respond to the processing of anti-DEC and control non-specific antibodies It is assessed by analyzing specific T cell proliferation.   The structure of the DEC of the present invention can be analyzed by various methods known in the art. . Analyze structures of various domains, especially lectin binding domain and cytoplasmic domain Is preferably performed. Structural analysis determines sequence similarity to other known proteins Identification can be performed. The degree of similarity (or homology) is DEC, or So Can provide a basis for predicting the structure and function of the domain of For special embodiments In the sequence comparison, for example, the FASTA program and the FASTP program (Pearson program) And Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444-48). Can be performed using the sequences found in   Protein sequences were analyzed for hydrophilicity (eg, Hopp and Woods, 1981, Proc. Natl. Acad. .Sci.U.S.A. 78: 3824). Hydrophilic profile is DEC It can be used to identify hydrophobic and hydrophilic regions of a protein.   Also, secondary structure analysis (eg, Chou and Fasman, 1974, Biochemistry 13: 222). ) Can be used to identify regions of the DEC that adopt a particular secondary structure.   Manipulation, translation and secondary structure prediction, reading frame prediction and plotting Use computer software programs available in the industry Can be achieved.   By providing a rich source of recombinant DEC, the present invention provides Enables quantitative structure determination of domains. In particular, enough material is nuclear magnetic resonance (NMR) Ready for infrared (IR), Raman, and ultraviolet (UV), especially circular dichroism (CD) spectroscopy You. In particular, NMR gives a very strong structural analysis of the molecules in solution, which A closer approximation of the natural environment (Marion et al., 1983, Biochem. Biophys. Res. Comm. 113: 967). -974; Bar et al., 1985, J. Magn. Reson. 65: 355-360; Kimura et al., 1980, Proc. Natl. A cad. Sci.U.S.A. 77: 1681-1685). Also, other methods of structural analysis may be used . These include X-ray crystallography (Engstom, A., 1974, Biochem. Exp. Biol. 11: 7-13). But not limited thereto.   It is further noted that the co-crystals of DEC and DEC specific ligands can be studied preferable. The eutectic analysis gives detailed information about the binding, which in turn Allows rational design of gonists and antagonists. Also computer Modeling can be used especially in conjunction with NMR or X-ray methods (Fletterick , R. and Zoller, M .; Editing, 1986, Computer Graphics and Molecular Modeling, C urrent Communications in Molecular Biology, Cold Spring Harbor Laborator y, Cold Spring Harbor, New York).   In yet another embodiment, the putative DEC of the present invention comprises It can be tested to determine if it cross-reacts with a specific antibody. For example, estimate DEC was reacted with a rabbit polyclonal antibody as described in the Examples below. To determine if it binds. In addition, DEC protein Which can be used to produce DEC-205 from mouse or human sources. One can test for differential reactivity. The degree of cross-reactivity depends on the structural homology of the protein or Gives information about similarities.   DEC carbohydrate composition includes lectin binding, chemical analysis, immunoassay, immunization Analysis (e.g., sugar conjugates are converted to digoxigen after periodate oxidation of vic-diol). Deglycosylation, enzymatic deglycosylation) Silation and exoglycosidase digestion followed by FACE (phosphor-assisted carbohydrate electricity Various means known in the art, including, but not limited to, Can be studied by   DEC ligand   Most importantly, the present invention relates to DEC ligands, such as DEC lectins. Advantageously, the identification of carbohydrate ligands binding to one or more of the mains is provided. This Such ligands are particularly useful for targeting binding to DEC. Book As used herein, “ligand” has its ordinary meaning, ie, the receptor, In some cases, it has a molecule that can specifically bind to DEC. Used herein The term "carbohydrate ligand" can specifically bind DEC Represents carbohydrate or sugar. Generally, also "gricazo", "saccharide", or Such carbohydrates, called "oligosaccharides," are carbohydrates of glycoproteins. Object part.   Identification and isolation of the gene encoding the DEC of the present invention can be accomplished by classical receptor binding. Transfection or transformation of cells in or for recombinant experiments Indicators specially manipulated to indicate the activity of the expressed receptor after exchange -In cells, the receptor, or its amount, in a larger amount than can be isolated from natural sources Gives expression of the truncated portion of Therefore, the present invention is known in the art. Various screening assays can be used to identify specific ligands for DEC. And intended. The recombinantly expressed protein may contain one or more DEC lectins. Main protein and can be truncated forms of the native protein or another protein. It may be a part of the native protein expressed as a chimeric construct with the protein. No.   All screening techniques known in the art are screened for DEC ligands. Can be used for training. The present invention relates to small molecule ligands or ligand analogs and mimetics. Screens, as well as screens for natural ligands that bind to DEC in vivo Intended. In particular, the present invention relates to the identification of carbohydrate groups that bind DEC, In particular, carbohydrate groups that bind DEC with high affinity and high specificity Provides the identification of   In a preferred aspect of the invention, the detection of the DEC ligand comprises the solubilized DE The C or DEC fragment can be conjugated to a sugar or sugar bound to a solid support such as Sepharose Is performed by binding to a column prepared from glycans (Taylor et al., 199). 2, J. Biol. Chem. 267: 1719). In particular, the present invention provides for only one or a few of the domains Various lectins by expressing truncated mutant DEC proteins It is intended to analyze the ligand specificity of the domain. Also, candidate glycans Is a carrier protein, such as bovine serum albumin (e.g.,¹²⁵I ) And cells, for example, recombinant cells as described above (Taylor et al., The binding to DEC or DEC fragment expressed by obtain. In yet another embodiment, labeled glycan-carrier protein binding Microtiter assay as described (Taylor and Drickamer, 1993, J. Bio l. Chem. 268: 399). Mannose as a candidate carbohydrate ligand , Fucose, N-acetyl-glucosamine, glucose, galactose, N- Acetyl-galactosamine, and a few other such carbohydrates , But is not limited to this. Other candidate ligands include disaccharides and larger Leader polysaccharides, such as those recognized by various lectins. You. As used herein, the term "detecting binding" refers to a type of association with another molecule. Commonly used to detect the association of molecules, eg, the association of DEC ligand with DEC. Represents all of the miriad technologies used. As these technologies, The immunoassay techniques described below, or improvements thereof, include Binding entity, polypeptide, to another entity, which is found on a solid support or cell The association of a label conjugated with DEC-lectin or one of the candidate ligands containing It depends on getting out. However, detection of binding, for example, This can be done indirectly by detecting the absence of a cognitive object. In another aspect Conjugation first removes unbound material and then removes labeled entities from the binding pair (Eg, using a chaotropic agent). To another object These and other techniques for detecting binding of a single object are publicly available in the art. Is knowledge.   For solid-phase or heterogeneous-phase assays, one member of the binding pair is solid-phase Body, eg, beads (eg, Sepharose), latex particles, chromatographs Fee carrier, magnetic particles, silica particles, silicon wafer, or plastic Will be irreversibly associated with the Rotaita plate. “Meeting irreversibly” The term non-dissociating or an assay time for which it is not visually detectable Represents a covalent or non-covalent bond characterized by a relatively low rate of dissociation.   Knowledge of the primary sequence of DEC, and the similarity of that sequence to carriers of known function, Provides early clues as inhibitors or antagonists of proteins Can be In this case, the macrophage mannose receptor and muscle The correlation of the deduced sequence of DEC with the sequence of phospholipase receptor is Aided characterization of DEC as a receptor with the tin domain. Antagonis Identification and screening are performed, for example, by X-ray crystallography, neutron diffraction, nuclear magnetic resonance Using spectrometry and other techniques for structure determination described above, It is further facilitated by measuring the structural features of the protein.   In a specific embodiment, the identification of the carbohydrate ligand of the DEC comprises the known glycan A classic neoglycoprotein, bovine serum albumin, or ovalbumin, or Or by attaching to a protein such as creatinase. In particular, it is advantageous to use proteins that are not naturally glycosylated. As a result, only the effect of the added glycan arc is analyzed. Binding assay above May include a classical binding assay as described above, or may comprise antigen-specific T lymphocytes May involve an antigen processing assay by assessing the stimulation of the antigen. to this In this regard, a number of T cell lines and clones specific for BSA and ovalbumin are available. Can be used. Neo-glycosylated BSA that effectively stimulates specific T cell proliferation or Ovalbumin's ability to conjugate to BSA or ovalbumin that binds to DEC Is an indicator of the ability of the glycans to be used.   In another embodiment, the heavily glycosylated, present in fetal bovine serum. Protein fetuin can be used to evaluate glycan ligands. T thin Develops a fetuin-binding system based on cell activation or marker endocytosis I can do it. Certain glycosidases specifically "knock-out" glycans And the ability of the modified fetuin to function in the binding system can be assessed. Function Loss of sex indicates that the enzyme-modified sugar residue was involved in binding to DEC. Would.   In yet another aspect, the 9th and 10th lectin domains of the DEC are antibodies. Membrane-associated antibodies by "chaperoning" the DNA into endosomes The observation that these domains may be involved in mediating antigen presentation suggests that these domains may be Suggests that it is specific for binding the carbohydrates found in the brin molecule.   Another approach is to use recombinant bacteriophages to build large libraries. Occurs. "Phage method" (Scott and Smith, 1990, Science 249: 386-390; Cwi rla et al., 1990, Proc. Natl. Acad. Sci. 87: 6378-6382; Devlin et al., 1990, Science 2 49: 404-406) can be used to construct very large libraries (10⁶-Ten⁸Of Object). The second approach mainly uses chemical methods and the Geysen method (Geyse n et al., 1986, Molecular Immunology 23: 709-715; Geysen et al., 1987, J. Immunolog. ic Method 102: 259-274) and a recent method by Fodor et al. (1991, Science 251, 767-77). 3) is an example. Furka et al. (1988, 14th International Congress of Biochemi stry, Volume 5, Abstract FR: 013; Furka, 1991, Int. J. Peptide Protein Res. 37: 487 -493), Houghton (U.S. Pat. No. 4,631,211 issued December 1986) and Rutt (U.S. Pat. No. 5,010,175, issued Apr. 23, 1991) discloses an agonist or agonist. Or methods of producing mixtures of peptides that can be tested as antagonists or antagonists. ing.   In another aspect, synthetic libraries (Needels et al., 1993, "Generation and s creening of an oligonucleotide encoded synthetic peptide library ”, Proc Natl. Acad. Sci. USA 90: 10700-4; International Patent Publication Nos.WO 92/00252 and 1 by Lam et al. U.S. Patent No. 5,382,513, issued January 17, 995, each of which is incorporated by reference. And the like) are used for screening for the ligand of the present invention. Can be used for   Also, one or more ligand binding domains (preferably multiple domains) of the DEC ) Was performed to determine the binding of soluble ligand to cells expressing the recombinant obtain. As described in the examples below, the presence of multiple lectin domains of DEC Location can contribute to the affinity and specificity of binding to glycans.   Screening can be performed using recombinant cells expressing DEC, or, for example, This can be done using a purified recombinantly produced receptor protein as described above. An example For example, a labeled, soluble, containing ligand binding portion of a molecule that binds a ligand Or the ability of the solubilized DEC to be used in a library, as described in the above references. And can be used to screen for   DEC antibody   According to the present invention, DEC produced by recombinant or chemical synthesis, and its Fragments or other derivatives or analogs, including fusion proteins, It can be used as an immunogen to produce antibodies that recognize C. With such antibodies Polyclonal antibody, monoclonal antibody, chimeric antibody, single chain antibody, Fa b fragments and Fab expression libraries, including but not limited to Absent. In a particular embodiment described below, the rabbit polyclonal antibody is DEC-205 with N Prepared for terminal amino acid sequence. In another embodiment, intact purified DEC-20 Five polyclonal antibodies were produced.   Various operations known in the art include DEC, or derivatives or analogs thereof. Can be used for the production of polyclonal antibodies. Rabbit, mouse Various host species, including, but not limited to, horses, rats, sheep, goats, etc. The product is a non-homogeneous DEC or a derivative thereof (eg, a fragment or fusion protein). Can be immunized by injection with (protein). In one embodiment, the DEC or its The fragment can be an immunogen carrier, such as bovine serum albumin (BSA) or Can be conjugated to keyhole limpet hemocyanin (KLH). Various adjuvants , Depending on the host species described above, may be used to increase the immune response.   Mono derived from DEC or a fragment, analog or derivative thereof For the preparation of clonal antibodies, the production of antibody molecules is provided by a continuous cell line in culture. Any technology that can be used can be used. These include Kohler and Milstein (1975, Nat. ure 256: 495-497), as well as the trioma technology Technology, human B-cell hybridoma technology (Kozbor et al., 1983, Immunology Toda y 4:72), and EBV-hybridoma technology for producing human monoclonal antibodies Surgery (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss . Inc., pages 77-96), but are not limited thereto. Additional features of the present invention In embodiments, the monoclonal antibody uses a recent technology (PCT / US90 / 02545) And can be produced in sterile animals. According to the present invention, human antibodies may be used, Human hybridoma (Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 2026-2030 ) Or in vitro using human B cells with the EBV virus (Cote et al.). , 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96) Obtained by transformation. Indeed, according to the present invention, a suitable biological activity Of DEC specific mouse antibody molecules together with genes from human antibody molecules By splicing the gene, "chimeric antibodies" (Morrison et al., 1984, J. Bacteriol. 159-870; Neuberger et al., 1984, Nature 312: 604-608; Takeda et al., 198. 5, Nature 314: 452-454) can be used. This Such antibodies are within the scope of the present invention.   According to the present invention, techniques described for the production of single-chain antibodies (U.S. Pat. No. 6,778) can be employed to produce DEC specific single chain antibodies. Addition of the present invention A specific embodiment is a technique described for the construction of a Fab expression library (Huse et al., 1989, Science 246: 1275-1281) using DEC or a derivative thereof, Is a Rapid Production of Monoclonal Fab Fragments with Desired Specificity for Analogs? Enables easy identification.   Antibody fragments which contain the idiotype of the antibody molecule may be generated by known techniques. You. For example, such fragments are produced by pepsin digestion of antibody molecules. F (ab ')_TwoFragment, F (ab^')_TwoFragment disulfide bridge Fab that can be produced by reduction^'Fragments and antibody molecules to papain and Fab fragments that can be generated by treatment with a reducing agent are mentioned. It is not limited to them.   In the production of antibodies, screening for the desired antibody is a technique known in the art. Techniques such as radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "Ditch" immunoassay, immunoradiometric assay, gel diffusion press Pitin reaction, immunodiffusion assay, in situ immunoassay (eg, colloidal gold , Enzyme or radioisotope labeling), Western blot, precipitation , Agglutination assays (eg, gel agglutination assays, hemagglutination assays), complement fixation Assay, immunofluorescence assay, protein A assay, and immunoelectricity It can be carried out by a migration assay or the like. In one embodiment, the antibody binding is that of the primary antibody. It is detected by detecting the label. In another embodiment, the primary antibody is a primary antibody It is detected by detecting the binding of a secondary antibody or reagent to the antibody. Yet another In embodiments, the secondary antibody is labeled. Binding is detected by immunoassay. Many means are known in the art and are within the scope of the present invention. For example, DE To select antibodies that recognize a particular epitope of C, such epitopes Hybridomas generated for products that bind to the DEC fragment containing Can be analyzed. For the selection of antibodies specific for DEC from a particular species of animal, Positive binding with DEC expressed by or isolated from such cells And the absence of binding to DEC from other species. Binding to DEC Can be detected as binding to dendritic cells expressing DEC.   The above antibodies can be used, for example, in Western blotting, in situ For formation, measurement of its level in a suitable physiological sample, etc., the DEC station It can be used in methods known in the art for localization and activity. The antibodies of the present invention Advantageously provides for the detection and counting of human dendritic cells. Also, such an antibody, for example, , Can be used to isolate human dendritic cells by panning. Yet another embodiment To In this context, the antibodies of the present invention may be used to target molecules to human dendritic cells. UU. It will be appreciated that this is a considerable advantage. Kraa This is because the conventional antibodies cannot recognize human DEC.   Targeted to DEC, DEC activity, eg, endocytosis Can be produced. Such antibodies can be used to identify ligands. Can be tested using the assay described above. In a specific embodiment, the rabbit polyclo A null anti-DEC antibody targets the binding of DEC and is subjected to endocytosis, Effectively presented to immunoglobulin-specific T cells.   Targeting molecules to DEC   The present invention relates to immunomodulation, such as stimulation of T cell immunity, suppression of immunity or T cell immunity. Induction of lugi and the mechanism of clonal deletion; Transepithelial by delivery of molecules across the skin or from the bloodstream to the lumen of the lungs or intestines Advantages transport; and targeting of molecules to DEC for crossing the blood-brain barrier Give to. In particular, a ligand of the above DEC, or the above DEC (or its D Antibody reactive with the EC binding moiety is conjugated to a molecule targeted to DEC Is done.   Immunomodulation With respect to immunomodulation, the present invention is particularly directed to T cell-mediated immunization for vaccination. It both stimulates the epidemic response and induces tolerance, especially with regard to autoimmunity.   Stimulation of T cell immunity is induced by anti-conjugated to a DEC binding moiety (ligand or antibody). The antigen, eg, weak or poorly immunogenic antigen, to the T cells first It is performed by introducing it to the subject along with the factor that activates the dendritic cells to be presented. obtain. Dendritic cell activation is achieved by using an adjuvant, such as the adjuvant described above (which is commonly used). (Which has the ability to induce a universal immune response). In addition, the “ Vaccines "are antigens conjugated to a DEC binding moiety and cytokines or lymphokines E.g., granulocyte-macrophage colony stimulating factor (GM-CSF), or May contain some other CSF. Antigens suitable for use in such vaccines Include bacterial, viral, parasite, and tumor antigens You.   The invention also provides for induction of tolerance. Tolerance is a deleterious immune response, especially Desirable to avoid autoimmunity and allograft rejection. For example, skin and lymph Presentation of antigen by non-activated dendritic cells in the T-cell region of the T-cell organs , Probably resulting in degradation of the responding clone. Thus, in one embodiment, Thus, tolerance is a condition under conditions that promote dendritic cell stasis, for example, in the absence of infection. Without the use of sorbents, and using a pyrogen-free pharmaceutical carrier, additional Modified by conjugation with a DEC binding moiety in the absence of a mhokine or cytokine It is induced by administering the given antigen.   Furthermore, it appears that high levels of DEC expression could act as a tolerizing effect. It is. Therefore, furthermore, the present invention provides a method for recombinantly administering an antigen conjugated to a DEC binding moiety. Recombinant modification to express dendritic cells or both DEC and MHC class II Transfer of the obtained cells to a subject. Also, the dec gene is To suitable cells in vivo.   In yet another embodiment, tolerance can be induced by a clonal deletion mechanism. You. In particular, antigens conjugated to the DEC binding moiety are mediated by thymic epithelium and spinal cord dendritic cells. For processing and presentation by targeting or physical injection It can be introduced directly into the examiner, preferably into the thymus. This processing and presentation process Involved in the selection process to eliminate autoreactive T cells, ie, clonal deletions It is considered to be. In yet another aspect, the level of DEC expression is, for example, Engineered by introducing additional dec genes into thymic epithelium and spinal cord dendritic cells. obtain.   DEC-research to induce tolerance, T cell anergy, or clonal deletion Attractive candidates for conjugation with Gand are allergens, autoantigens, e.g. Erin basic protein, collagen or its fragment, DNA, nuclear protein Protein and nucleolar proteins, mitochondrial proteins, pancreatic β-cell proteins But not limited thereto (Schwarz, 1993, Fundamental  Immunology, 3rd edition, edited by W.E.Paul, Raven Press, Ltd .: New York, 1033-1097 See page.)   Transepithelial migration In another embodiment, the molecule conjugates it to a DEC binding moiety It can be targeted for transepithelial migration. In one aspect of the present invention Thus, the present invention relates to the targeting of therapeutic molecules for absorption across the lung or intestinal epithelium I will provide a. Thus, the present invention provides for aerosol application by inhalation, ie, pulmonary administration of a drug. Providing delivery of the therapeutic agent used. In another aspect, the present invention provides a small intestine Provides delivery of therapeutic agents by DEC-mediated absorption across. In particular, the present invention relates to hydrophilic components. Provide for the absorption of, or more precisely, transmucosal translocation, these molecules are usually hydrophobic It is not as easily absorbed as the sex molecules. This aspect of the invention relates to the epithelial cell tip (or Or the presence of DEC on the surface (of the lumen).   In another aspect of the invention, the presence of DEC on the basolateral surface of epithelial cells To provide a pathway for the transport of DEC-ligand conjugated molecules to the lumen of the lung or small intestine I can. This delivery route is critical for the administration of acid labile hydrophilic therapeutics to the intestine. obtain. Such drugs cannot be taken. This is because the acidic conditions present in the stomach This leads to decomposition of Transport of such drugs from the bloodstream to the lumen of the intestine is automatic. It will not happen easily. What happens when hydrophilic drugs cross cell membranes It does not have a significant distribution coefficient. In a particular embodiment, the present invention provides Chemotherapeutics and antibiotics, especially antiparasitic drugs, are conjugated to DEC ligands and The agents are given by parenteral administration, preferably by intravenous administration, As a result, the drug is targeted for transport from the basolateral surface of the intestinal epithelium to the luminal surface. Is set.   Similarly, therapeutic agents target DEC receptors to the basolateral surface of lung epithelium. Can be targeted for delivery from the bloodstream to the lung airways . Such a delivery system would result in individuals with impaired lung capacity, for example, Thus, it would be particularly advantageous for delivery of the drug to an individual indicated for administration by blood flow. This Pulmonary dysfunction such as pneumonia, pus, lung cancer, adult respiratory distress syndrome, respiratory distress Difficulty, hemoptysis, chronic obstructive pulmonary disease (COPD), fibrosis-induced dust, pulmonary fibrosis, organic powder Dust, chemical trauma, smoking trauma, thermal trauma (burn or freeze), asthma (allergy , Bronchoconstriction, other causes of asthma, eg, irritants), hypersensitivity pneumonitis, Good These include Pasteur syndrome, pulmonary vasculitis, and immune complex-related inflammation. It is not limited to. Thus, the present invention relates to antibiotics for transepithelial transfer to the lumen of the lung. , Anti-flame Provides administration of anti-inflammatory drugs, complement inhibitors (eg, complement receptor 1 [CD35]), etc. I do.   In another embodiment, the blood-brain barrier migration is targeted to the brain The molecule can be conjugated to the DEC binding moiety. The molecule is then found in the brain capillaries D It is thought that they bind to EC and promote trans-brain barrier transport or migration. You. Currently, there are few mechanisms for guiding molecules across the blood-brain barrier. Not, or generally not valid. For transport across the blood-brain barrier Such molecules as neurotrophic factors (brain-derived neurotrophic factor, NT-3, NT-4, ciliary body Neurotrophic factor), growth factors (eg, nerve growth factor), etc .; for early infection of the brain Antibiotics or antivirals; and vectors for gene therapy But not limited to these.   Vector targeting for gene therapy In yet another embodiment The present invention relates to cells expressing DEC, especially dendritic cells, thymus, small intestine and lung. Provides ligands for targeting DNA vectors to skin and brain capillaries Offer. Therefore, DNA vectors such as viral vectors express DEC It can be modified by conjugation with a DEC ligand for targeting to cells.   As an example of a DNA virus vector, a defective herpesvirus 1 (HSVI) vector (Kaplitt et al., 1991, Molec. Cell. Neurosci. 2: 320-330), attenuated adenovirus Vectors such as Stratford-Perricaudet et al. (1992, J. Clin. Invest. 90: 626-6 30), a defective adeno-associated virus vector (Samulski et al.). , 1987, J. Virol. 61: 3096-3101; Samulski et al., 1989, J. Virol. 63: 3822-3828) , And papilloma virus vector, Epstein Barr virus (EBV) vector, And the like, but are not limited thereto. Virus particles are, for example, Chemically cross-link Gand to virus to include DEC ligand Can be modified to   Also, the vector can be introduced in vivo by lipofection. Ribosome medium Designed to limit the difficulties and dangers encountered in transfection In vivo transfection of DEC-encoding gene by synthetic cationic lipid (Felgner et al., 1987, Proc. Natl. Acad.Sci.U.S.A. 84: 7413-7417; Mackey et al., 1988, Proc. Natl. Acad. Sci. U.S.A.8 5: 8027-8031). Use of Cationic Lipids Encloses Negatively Charged Nucleic Acids Promotes fusion with negatively charged cell membranes (Felgner and Ringol d, 1989, Science 337: 387-388). Introducing exogenous genes into specific organs in vivo The use of lipofection to perform has certain practical advantages. Specific details Molecular targeting of liposomes to vesicles represents one area of benefit. It Therefore, the present invention provides a method for conjugating a DEC ligand to a liposome vector. It provides for targeting genes to cells and thymic epithelium. Lipids are tar Can be chemically coupled to other molecules for targeting purposes (Macke y et al., 1988, supra). Targeted antibody or glyca Can be chemically coupled to liposomes.   This vector can be introduced in vivo as an exposed DNA plasmid. And preferably using DEC ligand as vector transporter (Wu et al., J. Biol. Chem.,267:963-967 (1992); Wu and Wu, J. Biol. C hem.,263:14621-14624 (1988); Hartmut et al. Filed March 15, 1990. Nada Patent Application No. 2,012,311).   The present invention will be better understood by reference to the following examples. However, these are provided by way of example and not limitation. Example 1 Murine Dendritic Cells Detected by Monoclonal Antibody NLDC-145 -205 kDa protein, DEC-205, abundant in thymic epithelium and thymus epithelium: purification, characterization, And N-terminal amino acid sequencing   This example demonstrates the purification and purification of the antigen recognized by monoclonal antibody NLDC-145. And biochemical characterization (Krall et al., J. Exp. Med.163:981 pages (1986)). We use this protein in dendritic and thymic epithelial cells (Dendritic and  thymic Epithelial Cells) And observed as a molecular mass. This protein is of a scale that allows direct biochemical research Has been purified. This antigen is a weakly alkaline (pI7.5) integral membrane glycoprotein. And a molecular weight of 205 kDa by electrophoresis, which was initially reported at 145 kDa (Kraal It is not shown in the above-mentioned paper). About 7 kDa of this mass Covalent bonds contribute. Using a panel of plant lectins, these glycans Preliminary information on the structure was obtained. Then, these glycans are added to various exoglycosides Digestion and electrophoresis (FACE) using fluorophores (Jackson's dissertation, Bioc hem J.,270:705 (1990); Jackson and William, Electrophoresis. ,12: 94 (1991); Jackson's dissertation, Biochem. Soc. Trans,twenty one: 121 (1993); Jacks on, Anal. Biochem. ,216:243 (1994). Eight different but related two The branched N-linked glycan structure was analyzed. These variants have their ends Different, but based on two fucosylated trimannosyl chitobiose core structures (With and without bisecting GlcNAc). O-linked glycans are detected Was not done. The amino terminus of this protein is unblocked and The 25 amino acid sequence of is apparently not homologous to any known protein. And not similar. Two novel polyclonal antibodies have been described, Antibodies against the N-terminal peptide sequence and second against the complete purified protein Occurs. In immunoblots, both of these polyclonals were 205 kDa A band was observed, which was due to the preclearing of the extract with NLDC-145. It can be significantly reduced specifically.Materials and methods NLDC-145 Purification and preparation of immunoaffinity resin -NLDC-145 (rat IgG2a) belly Water was added to normal, 6-8 week old non-SPF, CD2 (BALB / c × DBA / 2F1) female mice (Tordo (Tr udeau) institute) in the manner described (North and Izzo discussions) Sentence, J.Exp.Med.,177:1723 (1993)). This monoclonal is immobilized Chromatography on Protein A and Protein G (Pierce) And purified. This monoclonal and non-specific rat IgG2a (dim Zymed) were both attached to the resin by reductive amination (AminoLi nk, Pierce).Immunoprecipitation -Bone marrow dendritic cells (BMDCs) were obtained using the method described by Inaba et al. (J. Exp. Med.176: 1693 (1992)), prepared from proliferating bone marrow precursors. 8 days after starting culture Eyes, BMDC 4.8 × 10⁷In a 10 ml medium without methionine and cysteine Cultured for hours. (³⁵(S) Methionine-cysteine (ICN) After labeling was started and after culturing for 4 hours, cells were collected. Ice cooled BMDC Lysis buffer (50 mM Tris-HCl, pH 6.8, 1% Nonidet P-40 ( Calbiochem), 50 mg / ml BSA (Intergen), In addition, the following protease inhibitor mixture: 5 mM EDTA, 0.5 mg / ml Pefabloc (Pef abloc) SC (Boehringer Mannheim), 100mg / ml PMSF, 5mg / ml aprotinin , 5 mg / ml pepstatin A and 10 mg / ml leupeptin (the last four are Sigma) 700 Dissolved by resuspending in ml. Lysate for rat IgG (jacko 20mg, FCS 10ml, and packed immobilized protein G1 Precleared with 0 ml (Pierce). This supernatant is mixed with 100 ml of protein G for 1 hour. A second pre-clearing was performed. Add 100 ml of the clarified lysate Divided into recoats. Protein in two aliquots (6.9 x 10⁶BMDC equivalent) , Filled and washed immunoaffinity resin 50 ml (NLDC-145 or rat IgG2a ) And rotated for 1 hour. Wash as described (Firestone and Winguth, Methods Enzymol.182:688 pages (1990) ). Proteins were analyzed by SDS-PAGE using a 10% acrylamide minigel.   Immunoblot method-Convert SDS-PAGE to 1.5% thick 8% acrylamide mini gel I went there. Nitrocellulose (BA-85, Schleich transfer was performed at 4 ° C. overnight at a constant pressure of 30 V. H Filter with 3% w / v nonfat dry milk and tweed Blocked for 1 hour at room temperature with shaking in 0.1% PBS containing 0.1%. one Secondary antibody (purified IgG 0.1-10 μg / ml, ascites or serum diluted 1: 1000, or diluted 1: 1 Incubated with the diluted hybridoma supernatant) in a heat-sealed bag. Performed for 1 hour at warm. Wash the filter and then peroxidase-F (ab ')_TwoDonkey Immunostaining using anti-rat or anti-rabbit IgG conjugate (Jackson) And subsequently enhanced with chemiluminescence (Amersham).   Purification of DEC-205 from thymus-The method is summarized in FIG. 50 per preparation Thymus from CD-1 Swiss mice (Taconic) outbred did. Thymus was placed in 50 ml ice-cold PBS containing 200 mg / ml PMSF and 5 mM EDTA. The blood was removed and washed once with the same buffer. -20 washed organs Can be frozen at ℃. All subsequent purification steps were performed at 0-4 ° C. Thymus To a 40 ml Dounce homogenizer (Kimble / Kontes) And a hypotonic lysis buffer (10 mM Tris-HCl, pH 6.8, mixed with protease inhibitors) Add substances: 5 mM EDTA, 100 mg / ml PMSF, 4 mg / ml aprotinin, 0.5 mg / ml pefab Rock SC, 4 mg / ml pepstatin A, 10 μg / ml leupeptin) resuspended in 30 ml . The organs were homogenized in 20 strokes with loose inner sections (gap 0.2 mm), and then Homogenized with 20 strokes of a tight inner cylinder (gap 0.1 mm). This suspension is It was left on ice for 20 minutes and then homogenized again with a tight inner cylinder for 20 strokes. Nuclear And debris were pelleted by low speed centrifugation (1200 × g, 5 minutes, 4 ° C.). Collect the opaque “postnuclear” supernatant and pellet the nuclear pellet with a hypotonic lysis buffer. 15 ml of buffer was changed 5 to 8 times, and the supernatant was washed until almost clear. The supernatant of each wash was combined. After harvesting, the nuclear supernatants were collected for 100, Centrifuged at 000 xg for 1 hour at 4 ° C (RC-28S centrifuge, F28 / 36 rotor, Upon-Solvall). The protein in the membrane pellet contains 0.5% (8.3 mM) NP-40. And extracted in 5 ml of hypotonic lysis buffer. This membrane extract was used for a second 100,000 × g, clarified by centrifugation for 1 hour.   The clarified membrane extract was precleared through a non-specific rat IgG column. . Fractions not adsorbed to the pre-clearing column are combined and then the NLDC-145 Added to the Nity column. Washing is performed in two steps: washing 1 is 6 ml (adsorbent bed volume) (0.5 times NaCl, but not NP-40, 0.5% (17 mM) n-octylglucose With hypotonic lysis buffer, replaced by Sid (Boehringer Mannheim); washing 2 (wash 1 without NaCl) was performed in 10 ml. This column is 50 times the bed volume mM glycine-NaOH, pH 11, 0.5% n-octyl glucoside, maximum flow rate 10 ml / cm^Two / Hour and eluted. The eluted fraction (1 ml) was added to 2-30 g of 2M glycine-HCl at pH2. The pH was adjusted to 7 using l. Combine the peak eluates against the plastic Ultrafiltration Centri precoated with 0.1% SDS to reduce non-specific losses It was concentrated to less than 1 ml using a con-100 unit (Amicon). Typically, From 50 thymus, 70-150 μg of DEC-205 could be obtained.   Isoelectric focusing-Perform isoelectric focusing on a thin (0.75 mm) slab gel under denaturing conditions. (8M urea, 4% all ampholine(pH 3.5-10 and 5-7 ratio 2: 1, Pharmacia), 0 5.5% acrylamide gel containing .67% NP-40, 10% glycerol) . The sample converges overnight at a constant pressure of 400 V at a minimum of 6000 V-hours, when the current is less than 1 mA. Was. Rows are silver-stained or cut into 0.5 cm sections for pH gradient measurements Then, degassed dH_TwoEluted in O.   Glycan detection-DEC-205, transferrin (positive control) and creatine Ze (negative control) was blotted on nitrocellulose as described above. Complex carbohydrates Is non-selectively periodate-oxidized to relax the vic-diol to form an aldehyde, It was converted to goxigenin (DIG) -labeled hydrazone. The staining pattern is alkaline Sphatase-anti-DIG-antibody complex (First CHOice, Boehringer Mannheim) Visualized with.   Chemical deglycosylation−DEC-205 (40 μg) and apotransferrin (100 μg , Positive control), 100 μl of each of the two samples was subjected to G-25 SF spin chromatography. Therefore, they were transferred to 0.1% trifluoroacetic acid, 0.05% SDS and then freeze-dried. Nothing Water trifluoromethanesulfonic acid (Sojar et al., Methods Enzymol.,138:34 (P. 1 (1987)) (Oxford Glyco System). Polype The peptide was separated from the cleavage product and the excess reagent was precipitated with TCA, followed by 8% Untreated controls were electrophoresed next to each other on an acrylamide minigel.   Enzymatic deglycosylation-Flavobacterium meningosepticum erium meningosepticum) -N-glycosidase (PNGaseF, Veri Glycan to which asparagine was bound was cleaved using Terentino et al., Biochem.,twenty four: 4665 (1985)). Aliquot of DEC-205 ( The final gel (10 μg per row) was denatured by boiling for 5 minutes in the presence of 0.1% SDS. After cooling on ice, spin briefly in a microfuge and collect the liquid, 5 × P NGase buffer (250 mM sodium phosphate, pH 7.0, 50 mM EDTA, 2.5% NP-40 5% 2-mercaptoethanol), then add 1 unit of PNGaseF. (5 μl) was added. Incubate the reaction overnight at 37 ° C, then 4x non-reducing Stop by adding 1/4 volume of SDS-PAGE sample buffer and boiling for 5 minutes. Stopped.   Lectin blot method-Plant lectins labeled with several digoxigenins (vegetable -Linger Mannheim) using DEC-205 and appropriate positive and negative controls. Glycoproteins were electroblotted and stained. Lectin used for staining , Its specificity and concentration are shown in Table 1.   Exoglycosidase digestion and FACE analysis-N-linked oligosaccharides are From the DEC-205 and the fluorophore ANTS (8-aminonaphthalene-1,3,6- (Sulfuric acid) (Jackson, supra (1990); Jackson and William). ms paper (1993); Jackson paper (1993); Jackson paper (1994)). Recombinant exhaust Soglycosidase was obtained from Glyco. Electrical fluorescence intensity is SE1000FACE word Visualized on a workstation (Glyco).   Amino acid sequencing-Thickness of DEC-205 prepared using Duracryl (Millipore) Electrophoresis was performed on multiple rows of 1.5 mm 4% minigel. Gel to polyvinylidene fluoride Blotted on den (PVDF, Bio-Rad). After transfer, filter 1% Immerse in acetic acid for 1 minute, stain with 0.1% Ponceau S for 2 minutes, then dH_TwoDecoloring quickly with O did. The 205 kDa band was excised and submitted for analysis. This N-terminal sequence is TP program (Altschul et al., J. Med. Biol.,215:403 pages (1990)) , BLAST Internet Services (NCBI, National Library of Medicine, NIH) And collated with all databases.   Polyclonal antibody to the complete DEC-205-2 New Zealand whites 4% of 1.5mm thick Coomassie-stained rabbits (Hazelton) The 205 kDa band excised from the Duracryl SDS-PAGE gel was injected six times. The dose is Per animal, per injection, in the range of 40-70 μg of stained protein (4- 6 sections), which were injected every three weeks, and injected with test blood (about 15 ml of serum). Collected two weeks after the firing. For the first injection, the sections were complete Freund's It was emulsified in Cubant (CFA) and injected subcutaneously at multiple sites on the back. Freund Complete adjuvant (IFA) was used as booster adjuvant. Response to serum tilt At oblique doses, crude thymus membrane extracts were Western blotted and followed. These animals The unfractionated eluate from the aforementioned immunoaffinity column, i.e. Additional boosts were made with soluble protein rather than gel slices. Flat per injection Average Four boosters of 50 μg were given to both rabbits. The IgG fraction was converted to protein A Prepared by chromatography.   N- Polyclonal antibody against terminal peptide-The method combined with hapten It focuses on a single cysteine at residue 19 (Figure 6A). Peptide N1 (SESSGND PFTIVHENTGKC) (SEQ ID NO: 2) was performed using maleimide chemistry (Imject, Pierce). To bind keyhole limpet hemocyanin (KLH) and ovalbumin (OVA). Was. On average, about 250 peptides are conjugated to each KLH molecule, with 6 pairs per OVA molecule. The peptide was complexed. This KLH-peptide conjugate was added to 400-500 μg each of Aliquot And two injections of 2 New Zealand White rabbits (200-2 50 μg / injection), again emulsified in CFA for the first immunization and in IFA for the booster. In order to remove anti-KLH activity from the serum, they are placed on a KLH-cysteine column. Pre-cleared. An anti-peptide antibody is conjugated to an unrelated carrier Isolated on a Tide-OVA column.result   145kDa Of NLDC-145 immunoprecipitation and Western blot of 205kDa antigen but notLaw-NL To determine the molecular weight of DC-145 antigen, DCs were used to expand bone marrow progenitor cells in vitro. (Inaba et al., Supra). This method uses the high purity NLDC-145 (+) DC. On day 8 of the culture, this DC was metabolized to form [³⁵S] Methio Labeled with Nin-cysteine. Extracts can be used for immobilized NLDH-145 or control non-specific Immunoprecipitation was performed using any of rat IgG2a. After reduction by SDS-PAGE (Fig. 1A) Perform autoradiography of these precipitates and determine the apparent mass of the NLDC-145. Binds to one specific band slightly larger than 200 kDa (myosin marker) But did not bind to the previously reported 145 kDa (Kraal et al., Supra). Revealed that. Prominent band at 45kDa, probably G-actin Numerous non-specific bands, including, were also visualized in both columns (Fosman, And Molecular Biology Handbook, Volume 1, Protein, CRC Press, Cleveland De, ohio).   To prove the results of this measurement, NLDC-145 was Used as Five thymus extirpated from 8-week-old BALB / c mice were subjected to immunoprecipitation. Homogenized in 2 ml of the same lysis buffer used. Transparent thymus extraction A gradient of the output is electrophoresed under non-reducing and reducing conditions and nitrocellulose Blot. NLDC-145 was transferred with a 205 kDa prestained myosin marker. Bound to one moving main band (Figure 1B), and immunoprecipitation estimates (described above) proved Was done. Under non-reducing conditions (left filter in Fig. 1B), only 7x10-^FourThymus The amount was clearly visualized at this dose of monoclonal NLDC-145 IgG. Only After the disulfide in the crude extract was reduced with mercaptoethanol (right On the side), this mAb can stain even at the highest dose of lysate. Did not. We note that the antigen recognized by NLDC-145 has a mass of 205 kDa and Concluded that the antigenic determinants detected on the mAb required intramolecular disulfide bonds .   DEC-205 Purification-Thymic cortex epithelial cells express large amounts of antigen (Kraal, supra). Et al.), The thymic medulla is slightly more abundant than DC. Therefore, from the thymus A method for isolating the protein has been developed (FIG. 2). Briefly, the chest The glands were treated with hypotonic lysis strips in the presence of six high concentrations of the protease inhibitor "cocktail". Homogenize under the subject. The membrane is isolated and extracted with NP-40. After clearing, Extract was precleared on a rat IgG column and then chromatographed on an NLDC-145 column. Rough.   Purified and concentrated SDS-PAGE analysis of DEC-205 (Figure 3A) was used for Coomassie staining. Therefore, a prominent 205 kDa band and a very small amount of contaminating band were revealed. Silver stain Reveals a large number of low molecular weight contaminants and can be analyzed before amino acid sequencing. Further purification was indicated by preparative electrophoresis.   In order to increase the yield of the next step in the purification step, the important fractions are (Volume of nuclear supernatant) and immunoblotted (FIG. 3B). This fraction is Therefore, the staining intensity on the filter reflects the relative concentration of DEC-205 in each separated phase. Should be reflected. Generally, in the first step of hypotonic lysis, 30 % Was apparently lost in the nuclear pellet (1 row). During the first ultracentrifugation, About 10% (3 rows) was lost to the precipitate containing the membrane pellet (4 rows). Constant small Fractions-probably 5-10% cumulative-are the early and late non-adsorbed fractions (rows 6 and 7) And passed through an NLDC-145 column. As a result, the cumulative yield of the final eluate (8 rows) The rate was about 50%.   If this eluate is intentionally overloaded by a factor of 5 at constant volume (column 10), At least six smaller sub-band “ladders” were clearly and consistently observed. Was. All of these bands should contain the NDLC-145 antigenic determinant. They are , Up to the 80 kDa component of considerable strength, in a neat form (ie, a continuous “spread”) (Not in shape). Carefully examine the initial fractions (especially rows 2, 3, 4 and 8) This reveals that these minor bands are present from the early stages of isolation. Or it becomes. Presumably, these bands represent the entire 205 kDa protein Would have been produced by pyrolysis.   DEC-205, an integral membrane protein with an isoelectric point of 7.5−DEC-205 protein, etc. To determine whether the protein is an integral membrane protein with a (Prepared in FIG. 2) was resuspended in the following solution: (1) hypotonic solution containing 0.5% NP-40 (2) same buffer containing 1M KCl instead of detergent (3) Contains a total of six protease inhibitors used for normal growth , 100 mM Na_TwoCO_Three, PH 11.5. After gently stirring for 1 hour, the suspension was clarified (100,000 × g, 60 minutes, 4 ° C.), and the supernatant was collected. Next, at high salt and high pH The precipitate obtained from the extraction was resuspended in a hypotonic lysis buffer containing 0.5% NP-40. And the above extraction and clarification steps were repeated. The five extractions generated in this way The Western blot of the product (FIG. 4A) shows that DEC-205 shows high ionic strength (2 Column) or extreme pH (3 columns) conditions do not release from membrane pellets. Revealed. A surfactant was required for its solubilization (1, 4 and 5 rows). Thus, DEC-205 is an integral membrane protein (Fujiki et al., J. Cel. l Biol.,93: 97 (1982)).   Isoelectric focusing was performed under denaturing conditions on a slab gel with silver staining. Relatively A uniform isoelectric point was observed at pH 7.5 (FIG. 4B). At this pH, a sharp convergence center bang Is spread out from pH 7.4 to pH 7.6 by a narrow "fringe" It was clearly bordered.   DEC-205, a glycoprotein that produces heterogeneous N-linked glycans−DEC-205 is To determine if glycosylated, the purified 205 kDa protein was Transferrin (a known glycoprotein) and creatinase (a glycoprotein The sample was also electrophoresed on a gel that was run, and electrophoresed on nitrocellulose. Was blotted. Filter at room temperature using sodium meta-periodate To convert non-selectively adjacent diols in the saccharide to aldehydes. Gigot Xygenin (DIG) -labeled hydrazide is applied to the filter and the aldehyde Was converted to DIG-hydrazone. Block this filter, then covalently bind DIG was stained with an anti-digoxigenin antibody-alkaline phosphatase complex and analyzed. Issued. This staining pattern (Fig. 5A) shows that DEC-205 (2 rows) (1 row) It was revealed that it was a similar glycoprotein.   Use this refinement to determine how glycans contribute to the apparent molecular weight. The produced protein was chemically deglycosylated. Anhydrous trifluoromethanes Rufonic acid (TFMSA) does not attack the primary structure of the protein but binds to asparagine. Both combined and serine / threonine-linked glycans are labeled with their amino acid side chains. Hydrolyzes at the site of the binding of (Cited by Sojar et al .; Dabich et al. hem.Biophys.Acta,1164: 47 (1993)). Apotra treated with TFMSA (Figure 5A) Both unferrin (2 rows) and DEC-205 (4 rows) were treated with untreated samples (1 and 3). Compared to (column), showed increased electrophoretic mobility. Of this processed sample As expected from linear regression analysis of migration distance, the deglycosylated apot Transferrin loses 5 kDa of apparent molecular weight (MacGillveray et al., J. Biol .Chem.,258:3543 (1983)) On the other hand, it was found that DEC-205 lost roughly 7 kDa. It became clear. This 7 kDa shift is due to the N-linked glycan of the 205 kDa protein. Of two or three complex types. Some smaller O-linked glycans Was removed.   To begin determining the type of carbohydrate residues present on this protein, The purified material was probed with a panel of digoxigenin-labeled plant lectins. A Treatment of spargin-linked glycans with peptide N-glycosidase F (PNGaseF) This was removed from the aliquot of DEC-205. Processed and unprocessed protein Quality was blotted onto nitrocellulose, along with glycoprotein positive and negative controls. Was. After staining the filter with PonceauS (not shown) and confirming metastasis, the membrane was blocked. And stained with DIG-lectin at the concentrations shown in Table 1.   The lectin staining pattern (Table 1) shows that N-linked glycans Lactin does not bind to SNA and MAA. N-Acetylneuramin bound to either α2-6 or α2-3, a ketose bond No acid was shown. Since the PNA did not bind, the O-linked glycan The core of the disaccharide is not present in the unsubstituted form. Due to the neuraminidase of this protein Pretreatment does not make the stain more susceptible to PNA (not shown); The presence of any O-glycans present was not covered by sialic acid. N-bonded grease Selective removal of cans by PNFGaseF reduces the apparent mass of this protein by 7 kDa These numbers, if any, are small and non-selective Only chemical deglycosylation occurs. Undigested DEC-205 is strongly stained by DSA And this staining is removed by PNGaseF digestion. Therefore, one of the N-glycans More than one species are terminated with Gal β1-4GlcNAc. Undigested protein by AAA Because it stains deeply, the terminal fucose linked to GlcNAc with α1-6 also Present in at least one species. Lectin GNA is more active than the DSA (+) and AAA (+) bands. Slightly stain the band that migrates quickly. Probably this 200kDa band is high It contains mannose-type N-linked glycans, which are still present in the Golgi complex. FIG. 3 represents a subgroup of newly synthesized molecules that have not undergone a glycose treatment reaction.   The structure of N-linked glycan of DEC-205 was further analyzed by carbohydrate electrophoresis (F ACE). PNGaseF is eight different N-linked glycans from DEC-205 The structure was released and the electrophoretic mobility was between 5.1 and 10.1 glucose units (Fig. 5C). The yield of this glycan is so low that each of these eight bands was cut. Extraction and sequencing were not possible, so this mixture was separated with exoglycosidase. (FIG. 5D). Digestion with α-galactosidase (2 rows) simplifies the pattern Which indicates that some glycans terminate in Gal α1- (1 or 2) Gal . NANaseIII (3 rows, α2-3, α2-6 and α2-8 linked N-acetylneuramin The addition of acid-specific further simplifies the pattern (loss of sialic acid causes band migration) Degree decreased. This means that some of these glycans are terminated by sialic acid. Was shown. NANaseI (selective for α2-3 binding) and NANaseII (α2-3 and α2-6 binding) (Selective for both cases) did not change the band pattern (not shown). this is Specific DEC-205 group, consistent with lack of staining with lectins SNA and MAA (described above) It was shown that the terminal sialic acid present on lican was an α2-8 bond. Further β- Galactosidase treatment (lane 4) resulted in loss of two galactoses (DSA A bifurcated structure corresponding to the staining), and the complexity of the pattern to the doublet is reduced Was. Addition of β-N-acetylhexosaminidase to this enzyme mixture (row 5) In addition, the small band of this doublet releases 2GalNAc and the fucosylated A band characteristic of the limannosyl chitobiose nucleus resulted. But the upper band “Dichotomous” GlcNAc that survived digestion and bound to the trimannosyl nucleus between the branches Or perhaps a fucose branch was present. The bisecting GlcNac is For double enzyme concentration (row 6), the upper band is at least partially digested (The fluorescence intensity was reduced by a factor of 2 or more). More α-man Digestion with nosidase (row 7) is incomplete and in most cases a single manno (Main band) but fucosylated mannosyl chitobiose nucleus A band that moves with the structure (9 rows "FC") is shown to be emitted. α- The amount of mannosidase was doubled, and the binding of α1-2, -3, -4 and -6 to GlcNAc By adding course specific α-fucosidase (row 8), substantially Moves with fully digested, non-fucosylated mannosyl chitobiose nucleus A band (9 rows "C") was released.   By combining lectin staining and FACE analysis, DEC-205 With two fucosylated core structures, with or without GlcNAc; It was found to contain biantennary N-linked glycans (FIG. 5E). Further heterogeneity Was introduced at the outer ends of these structures, which in all eight mutations were α-linked. Lactose, β1-4 linked galactose, or α2-8 linked sialic acid End.   N- Terminal amino acid sequence, and polymorphism of N-terminal peptide and complete DEC-205 Clonal antibody -Send the purified DEC-205 to amino acid terminal sequencing and The first 25 residues were clearly identified (FIG. 6A). This sequence is used for all available tamper No significant homology was found when compared to the protein sequence database . Our purified 205kDa protein is the antigen recognized by NLDC-145 To prove that, a 19-residue N-terminal peptide (Figure 6A, first 19 amino acids) Synthesized, purified, coupled to keyhole limpet hemocyanin, and Gi was injected. Peptide-reactive antibodies from hyperimmune sera were transferred to an unrelated carrier On an affinity resin prepared by binding the peptide to albumin Was purified. In parallel, purified DEC-205 was injected into a second rabbit pair, Hyperimmune IgG was purified by protein A chromatography. Immunoblo In the immunoassay method, both polyclonal antibodies, like NLDC-145, were The 5 kDa band was stained (FIG. 6A, “Extract” row). For both polyclonal antibodies Affinity for the loaded 205 kDa protein is greater than for monoclonal antibodies. It was 100 times higher. Here, 0.1 μg / ml of any polyclonal antibody was used for NLDC-1 The staining intensity was equivalent to that obtained with 10 μg / ml of 45 IgG. Both polychromes The primary antibody binds to the minor band “ladder,” similar to what is seen with mAbs. Was. Staining of the 205 kDa band indicated that the extract was precleared with fixed NLDC-145 In this case (Fig. 6B "depletion"), it is specifically removed but eluted from the pre-clearing resin. (Figure 6B "elution"), indicating that the correct protein Has been purified and sequenced.Consideration   Using a purification method based on immunoaffinity chromatography, mAbN An antigen bound to LDC-145 was isolated, and was isolated from mouse dendritic cells and thymus. It is reported to be expressed at high levels by the epithelium (Kraal et al., Supra). Sentence). As a protein source, we are trying to purify a large number of DCs. But the whole thymus of the mouse was lysed. This isolated protein has a purity of about 95 % And sufficient for N-terminal amino acid sequencing and basic biochemical testing. Yields in the range of μg were obtained. Nearly 10 to obtain an equivalent amount of protein⁹~Ten^TenD C will be needed.   The molecular weight of this protein by electrophoresis was consistently 205 kDa. Kraal In their original report (supra), NLDC-145 was used to reduce the density of iodized surfaces. Detergent extracts from lymph node cells were immunoprecipitated. Single serine protea Only the enzyme inhibitor 1 mM PMSF was present in the lysis buffer. One prominent mark Under both reduced and non-reducing electrophoretic conditions, the perceived protein Binding to 145 kDa, the author added the number "145" to the name of this clone. Pure When they tried to reproduce this immunoprecipitation (J.Exp.Med.,172:1459 (1990)) Number of serine protease inhibitors (1 mM PMSF, 1 μg / ml aprotinin and 0.1% DI FP) containing this lysis buffer.³⁵S) methionine labeled culture A surfactant extract of the epithelial DC (Langerhans cells) was prepared. They reduce Under conditions, proteins with an apparent molecular weight of more than 200 kDa were analyzed.   In this example, not only serine protease but also sulfhydryl and Bone marrow trees in the presence of high doses of inhibitors of paraginic acid and metalloproteases Immunoprecipitation of dendritic cell-derived antigens produces proteins with an apparent molecular weight of 200 kDa or more This is consistent with the paper by Pure et al. Western blot under non-reducing conditions using mAb Blot staining was performed, and a molecular weight of 205 kDa was independently calculated by this method. NLDC-145 immuno The protein eluted from the affinity column has a molecular weight of 205 kDa. The amino terminus of the protein is compatible with all other proteins in the current database. No significant homology or similarity was found. This 205 kDa purified protein To demonstrate that the quality is the antigen detected by NLDC-145, the N-terminal peptide and And polyclonal antibodies against the completely purified protein were prepared. Both polycs Lonal antibody stained the 205 kDa band on the immunoblot. This staining was performed at NLDC -145 can be removed with the extract pretreated by It showed that the protein was purified and sequenced. Therefore, it was purified here The 205 kDa protein has the confidence recognized by the NLDC-145 monoclonal antibody. It is an antigen that can be cut. We have Kraal, the smallest anti-proteolytic protection range The lysis conditions used in these papers (supra) allow for limited degradation of this protein I think.   We report high levels of this protein by its dendritic cells and thymic epithelial cells. The name "DEC-" indicating the expression of the bell and supplementing the previous estimate of the molecular weight by electrophoresis 205 ”. DEC-205 is an integral membrane glycoprotein, This yields a complex N-linked glycan with 2-3 bifurcations containing about 7 kDa in molecular weight. This These glycans are formed in two different core structures and further differ in their ends. , Resulting in eight variants, some of which contain sialic acid. However, the isoelectric point In electrophoresis, the isoelectric point of DEC-205 is slightly alkaline (pH 7.5), This indicates that this protein has relatively many base residues. This pI is quite Uniform: A narrow "fringe" of protein dilutes around the main pI However, it is only pH 7.4 to 7.6, which limits the charge heterogeneity. It reflects what was done. The large total molecular weight of this protein and the Considering the relative smallness, the detected sialylated glycan variants were Does not confuse the pI of the DEC-205.   DEC-205 is very sensitive to proteolysis. During amplification, a wide range of Not to inhibit the Rotase and to remove the cytosolic fraction after hypotonic lysis Otherwise, the yield will be very low. This proteolysis is clearly It is clear that it will proceed on a route that is not useful. High concentrations in our buffers High levels of antigen despite the presence of six protease inhibitors When blotted, we always range up to about 80 kDa and treat NLDC-145 antigens. Six to eight separate low molecular weight subband "ladders" containing the fixed group were observed. this The neat sequence of the proteolytic fragments was obtained from the crude thymic membrane extract (best shown in FIG. 4). 1B and 3B), the purified and ultrafiltered immuno It is also seen in the blot of each preparation leading to the affinity eluate (FIG. 3B). Principal “Fringes” stained around the electric point are probably these relatively large tanks. The proteolytic fragment would have been produced at least in part.   Large in size, not randomly distributed, and relatively protease-resistant The minor band “ladder” tells us that the complete primary structure of DEC-205 is more protea Multiple protease-resistant domains linked by an enzyme-sensitive connecting segment Suggests a modular structure with accompanying Example 2: Dendritic cells having 10 C-type lectin domains and involved in antigen processing DEC-205, a receptor expressed by alveolar and thymic epithelial cells   This example demonstrates that DEC-205 interacts with macrophage mannose receptor (MMR). The same, a receptor with 10 C-type lectin domains, and binds to carbohydrates, And that it is another related receptor that mediates endocytosis reported. Fractionation of DEC-205 was performed using monoclonal and polyclonal anti-DEC-205 Checked with antibodies. Dendritic cell DEC-205 is rapidly internalized through coated vesicles, In a multivesicular endosomal compartment that appears to be an MHC class II-containing vesicle essential for primary processing It was decided to be sent. In addition, rabbit anti-DEC-205 antibody is Effectively processed and presented to rabbit IgG-specific T cell clones. This In these experiments, DEC-205 showed that dendritic cells and thymic epithelial cells A novel antigen that can be used to present antigens captured from the extracellular space Suggest that it is an endocytic receptor.Materials and methods   Purification of dendritic cells-Dendritic cells obtained from a 7 day culture of bone marrow cells, as shown in FIG. The vesicles were isolated from a polyclonal rabbit anti-DEC-205-F (ab) '2 fragment and a 10 nm gold-labeled goat. Treated with anti-rabbit IgG and prepared for electron microscopy. 10 grids for each time point Test, photograph all cells labeled with gold, count gold particles, and use standard morphology Unobserved observers scored based on the biological criteria. The number of parentheses is , Represents the percentage of total gold particles scored for each plot.   Northern blotting method-For Northern blots, add 2 μg of mRNA to 0.8 The gel was electrophoresed on a% agarose formaldehyde gel. Transfer the sample to a nylon membrane And antisense RNA probes spanning nucleotides 3688-5200 of DEC-205 cDNA Coupled with the The blot was then removed and the loading control Using a glyceraldehyde-3-phosphate dehydrogenase probe as Hybridization.   electronic microscopeDendritic cells taken from a 7-day culture of mouse bone marrow Polyclonal anti-DEC-205-Fab'2 fragment of null anti-DEC-205; or biotinylated Incubate with 10 μg / ml of Noclonal NLDC-145 on ice for 30 minutes. I did it. Excess primary antibody was removed from RPMI-1640, 10% FCS, 0.02% NaN_ThreeAnd wash the cells three times Purified and removed. Each of these cells was then used as a 10 nm gold-labeled goat anti-rabbit. With either a 1: 5 dilution of IgG; or a 1: 5 dilution of streptavidin labeled with 10 nm gold Both were incubated on ice for 30 minutes. Excess secondary reagents are removed by the aforementioned washing did. The dendritic cells are then fixed at midnight with 2.5% glutaraldehyde or if Alternatively, the cells were incubated at 37 ° C. for a certain time before fixation, and prepared for electron microscopy.   Antigen presentation-Dendritic cells from a 7-day culture of bone marrow from 105 BALB / c mice were 5 48 hours with T hybridoma cells specific for 2R.50 rabbit IgG Cultured in Kate (Boom et al., J, Exp. Med.,167:1350-64 (1988)). Supernatant Was measured for IL-2 concentration using an HT-2 indicating cell line. 2R.50 Production of IL-2 by cells Raws were plotted on a logarithmic scale against antibody concentration in the culture. Error bar Indicates the standard deviation from the mean.Results and discussion   This example reports the molecular properties of the 205 kDa cell surface protein described in Example 1. Tells. Using an oligonucleotide probe based on the sequence of the protein Thus, 14 cDNA clones were transformed into three individual thymus and dendritic cell cDNA libraries. Obtained from. All these cDNA clones were derived from the same mRNA. DEC-205 cDNA Clones containing the putative 5 'end encode the N-terminal peptide of the DEC-205 antigen and This was preceded by a hydrophobic leader sequence consistent with the signal sequence. This The protein consists of 10 C-type lectin domains, a transmembrane domain and a cytoplasmic domain. (FIG. 7).   This composite cDNA contains all 29 unambiguous DEC-205 peptide sequences, A single 5.2 kDa open, encoding a 1,722 a.a protein with a molecular weight of 195 kDa It had a reading frame (FIG. 8). DEC specifications for both mouse and human Of particular note is the high degree of identity and similarity of the cytoplasmic domain sequences (FIG. 9). This cD 7.5Kb mRNA corresponding to NA is expressed at high levels in dendritic cells, thymus and lymph nodes And the corresponding pattern is the tissue staining with the NLDC-145 monoclonal antibody. Obtained by color (Kraal et al., Supra) (Figure 10).   The sequence of DEC-205 is compared to known peptides in the database and Dimannose receptor (MMR) (Taylor et al., J. Biol. Chem.,265:12156-6 2 (1990); Ezekowitz et al., J. Exp. Med.,172:1785-94 (1990)) Phospholipase A2 (PLA2) receptor in heron skeletal muscle (Lambeau et al., J. Biol. .Chem.,269:1575-8 (1994)), that of bovine pancreas (Ishizaki et al., J. Biol. Chem. ,269:5897-904 (1994)). Group VI  Defined as type C animal lectins (Drickamer and Taylor, Annu. Rev. Cell Biol. ,9:237-64 (1993)), all known members of this family Type I transmembrane with short cytoplasmic domain that mediates puter endocytosis (Ezekowitz et al., J. Exp. Med.,172:1785-94 (1990) ; Ishizaki et al., J. Biol. Chem.,269:5897-904 (1994); Taylor et al., J. Biol. Chem.,267:1719-26 (1992)). Outside of MMR protein family cells The part has a clearly cysteine-rich N-terminal domain, to which fibrone Kutin type II repeats and 8 Ca⁺⁺-Dependent sugar chain recognition domain (C-type CRD) (Taylor et al., J. Biol. Chem.,265:12156-62 (1990); Lambeau et al. , J. Biol. Chem.,269:1575-8 (1994); Ishizaki et al., J. Biol. Chem.,269 : 5897-904 (1994)). DEC-205 replaces the usual eight C-type CRDs with ten It branches off only from this pattern (FIG. 7). Group VI lectin cis The function of tein-rich regions and fibronectin repeats has not been determined. versus In contrast, this C-type CRD is a sugar chain binding domain ((Drickamer and Taylor Paper, Annu. Rev. Cell Biol. ,9:237-64 (1993)), and MMR and That both rabbit and rabbit PLA2 receptors bind to sugar chains (Lambeau et al. Statement, J. Biol. Chem.,269:1575-8 (1994); Drickamer and Taylor, Annu. ev.Cell Biol.,9:237-64 (1993)), with detailed experimental evidence. Rat serum Defined as C-type CRD for Northose protein and E-selection, Glycan contact residues (Drickamer's paper, Nature,360:183-86 (1992); Weis et al. , Science,254:1608-15 (1991); Weis et al., Nature,360:127-34 (1992 ); Graves et al., Nature,367:532-8 (1994)) is preserved in DEC-205. Not done. However, the nature of the sequence originally defined as “essential” for glycosylation also Absent in CRD of NKR1 and rabbit PLA2 receptor that binds strongly to ligand ( Lambeau et al., J. Biol. Chem.,269:1575-8 (1994); Bezouska et al., Na ture, 372: 150-57 (1994)), so additional functions for glycosylation by C-type CRD Must exist. CRD has also been found in over 100 other proteins. (Drickamer and Taylor, Annu. Rev. Cell Biol.9:237-64 (1993)), CRD in DEC-205 is associated with bovine and rabbit PLA2 receptor and MMR. (BPLA2 receptor and 34.6%, and And 36.7% identity with hMMR). In fact, DEC-205's CRDI-5 and 7-8, and other groups There is an ordered correspondence between CRDI-5 and 7-8 of the Group VI animal lectin, DEC-20 5 CRD6 most closely resembles CRD1 in another family member. Unusual CRDs 9 and 10 of DEC-205 are most closely related to CRDs 7 and 8 of other group VI lectins. Related and can occur during gene replication. At least two CRs in MMR D binds to mannose (Taylor et al., J. Biol. Chem.,267:Pp. 1719-26 (1 992)), and some CRD groupings indicate that the affinity of MMR for glycan ligands (Taylor and Drickamer, J. Biol. Chem.,26 8: 399-404 (1993)). The same mechanism is implemented by DEC-205 Used to increase both the affinity and diversity of carbohydrate binding.   This receptor function is based on monoclonal and polyclonal anti-DEC-205 antibodies. Were examined using a combination of The cytosolic domain of DEC-205 contains a conserved aromatic Group amino acids (FIG. 9, SEQ ID NOS: 1 and 6), which Findings that are related as part of motivation (Ezekowitz et al., J. Exp. Med.172: 1785-94 (1990); Ishizaki et al., J. Biol. Chem.,269:5897-904 (1994 Chen et al., J. Biol. Chem.,265:3116-3123 (1990); Collawn et al., Cell,63: 1061-72 (1990)) shows that this receptor is used for dendritic cells and thymic epithelial cells. Used to deliver various extracellular glycoprotein antigens to the intracellular antigen processing compartment. Suggest that it can be used. To test this theory, dendritic cell DEC-205 Gold-labeled monoclonal and polyclonal anti-DEC-205 antibodies conjugated to Fate was examined by electron microscopy in a time course experiment (FIG. 11). Two individual In experiments, monoclonal or polyclonal antibody complete or F ( ab) Similar results were obtained using the '2 fragment. At 0, cells are on the plasma membrane 95% of the connected gold particles were found. After heating this sample at 37 ° C for only 1 minute , 38% of the particles were found in coated vesicles (vesicles or pits). Crosslink for 20 minutes After that, 79% of the gold particles are characteristic of dendritic cells (Steinmann et al., J. Ex. p.Med.,149:1-16 (1979); Kleijmeer et al., J. Invest. Dermatol.103:51 6-523 (1994)) and contains MHC class II which is thought to be related to antigen processing Similar to vesicles (Amigorena et al., Nature,369:113-120 (1994); Qiu et al. Paper, J. Cell biol.,125:595-609 (1994); West et al., Nature,369:14 7-151 (1994); Tulp et al., Nature,369:120-26 (1994); Schmid and Jack son's dissertation, Nature,369:103-4 (1994)) in the multivesicular compartment (Fig. 11 and Table 2). Therefore, DEC-205 is rapidly and internally internalized by the coated vesicles. Antibodies bound to the selected receptor are delivered to the multivesicular endosomal compartment.   Determine if DEC-205 can deliver antigen to active antigen processing compartment Dendritic cells were treated with rabbit anti-DEC-205 antibody and rabbit IgG-peptidic Assay of T cell / MHC complexes on T cell clones (Boom et al., J. Exp. Med.,167:1350-64 (1988)). Negative controls were non-specific rabbit antibodies and B cells Rabbit antibody to IgG2a effectively presented by the system (Boom et al. Sentence, J.Exp.Med.,167:1350-64 (1988)). Dendritic cells serve as T cell clones Rabbit anti-DEC-205 antibody compared to non-specific rabbit antibody or rabbit anti-IgG2a antibody , Two digits more (Fig. 12). Therefore, DEC-205 is Is efficiently internalized and the bound ligand is Similar to B cell membrane-type immunoglobulin delivered to the intracellular compartment (Chestn ut and Gray, J. Immunol.,126:1075-79 (1981); Rock et al., J. Exp. Med.,160:1102-25 (1985); Lanzavecchia's paper, Nature,314:538-39 (1985 )).   In conclusion, dendritic cells and thymic epithelial cells express the novel receptor DEC-205. And this contributes to antigen presentation. The multiple lectin domain structures described above This receptor can be used by dendritic cells and thymic epithelial cells to generate various antigens. Used for carbohydrate capture and endocytosis and directing them to the antigen processing compartment Suggest that it can be used. Example 3: Expression of DEC-205 on dendritic cells of mouse leukocytes and other subsets   Previous studies by various groups have shown that mAb NLDC-145 has It has been shown to react mainly with epithelial cells of the thymic cortex. Shown in Example 1 Thus, this mAb is a 205 kDa integral membrane glycotan with a unique amino-terminal sequence. Rabbit polyclonal that recognizes DEC-205 and recognizes purified DEC-205 Lonal antibodies have higher affinity for the blotted antigen than the original mAb was there. In this example, both polyclonal and NLDC-145 antibodies were Used to reevaluate the expression and function of DEC-205 above. Cell fluorimetry High DCs from epithelium (Langerhans cells) and proliferating bone marrow progenitor cells While expressing levels (2-3 log fold) of DEC-205, freshly isolated splenic DCs , With two subsets, most (80%) with low levels of staining (≦ 1 log fold) The rest was moderate (1.5 log times). DEC-205 antigen determination The group is sensitive to trypsin but is not regenerated in culture. Resident and flame Symptomatic peritoneal macrophages, except for small amounts on thioglycolate-induced cells, Did not express the antigen. DEC-20 of B cells of spleen, lymph node, bone marrow, blood and ascites 5 The expression level was 10-50 times lower than that on BMDC. Bone marrow pro and pre -B cells did not express DEC-205. Polyclonal anti-DEC-205 When cells are injected into F1 mice, primary mixed lymphocyte reactions with DCs in vitro Neither reflex stimulation nor local graft-versus-host reaction in vivo was inhibited. Therefore , DEC-205 is more widely expressed on leukocytes than previously expected .   Kraal et al.'S paper (J.Exp.Med.,163:Monochromena described on page 981 (1986)) Antibody was used in mice treated with NLDC-145 IgG for a long time in vitro or at birth. Also failed to block any of the DC functions tested (Breel et al. , Immunol.,63: 331 (1988)). Unique tissue of antigen recognized by NLDC-145 Due to distribution and in the cell types (dendritic cells) where few restricted mAbs were identified Due to its abundant expression, DEC-205 was retested for cell specificity and potential function. Improved detection of DEC-205 using a novel polyclonal antibody (described in Example 1) , And tried to confuse the DC function.Materials and methods   mouse− (C57BL / 6 × DB) by Toldo Institute (Saranak Lake, NY) (A / 2) and (BALB / C XDBA / 2) F1 mice, and ((Hamamatsu, Shizuoka, Japan) C57BL / 6 × BALB / C) Mature (6-10 weeks old) bisexual, including F1 and BALB / C mice Of mice were tested.   Cell suspension-Immediately after removal from animals or 5% FCS, 50 μM 2-mercaptoeta After culturing in RPMI-1640 medium supplemented with phenol and 20 μg / ml gentamicin, The cells were tested. Tear the spleen, thymus and lymph nodes finely with forceps, or Further digestion with collagenase (Swiggard et al., Latest protocol in immunology, Co Editing by ligan et al., Green Publishing Association and Weil Interscience: NY, supplement 3 Edition, pages 3.7.1-11: Crowly et al., Cell Immunol. ,118:108 (1989)) Was obtained. Bone marrow cells are aspirated from the femur and tibia with a syringe and blood is punctured from the heart And collected in heparin. Dissolve all cell suspensions in 0.83% ammonium chloride Lysed in the solution and depleted red blood cells. Dendritic cells were obtained from three sources, each of: mouse Ear skin sheet (Schuler and Steinman, J. Exp. Med.,161:526 pages (198 5)), splenic low-density plastic adherent group (Saveggard et al., Supra; Cr, supra) owley et al.), and expanded myeloid progenitor cells enriched with rGM-CSF (Inaba et al. J. Exp. Med., 176: 1693 (1992)). Peritoneal cells are indigenous group or various inflammations Proteose peptone 3 days before, 4 days before Thioglycolate broth, 50 μg concanavalin A 2 days before, or 10 days 7 days before⁷ The surviving microorganism Mycobacterium bovis BCG was administered. After 1-3 days of culture, Several groups were tested. B cells in the lymph node suspension are B cell mitogens. Lipopolysaccharide (10 μg / ml LPS, derived from E. coli 0111: B4, Difco, Detroit, MI ), Anti-IgM + IL-4 (10 μg / ml goat F (ab ')_TwoAnti-mouse IgM (Jackson immunoreactor Chisha, West Grove, PA), and Dr. T. Sudo (Toray Industrial Research Institute, Kamakura, Recombinant mouse IL-4 50 U / ml obtained by courtesy of Japan) and CD40 ligand (CD40 L: Transcript with CD40L (obtained courtesy of Dr. H. Yagita (Juntendo Medical University, Japan)) In the transfected L cells, fix with 1% paraformaldehyde, and Washed twice and co-cultured 1: 1 with B cells. ). Skin dendritic cells Cultured as a skin suspension, then floated with concentrated bovine serum albumin (flotation) and concentrated (Schuler and Steinman, supra), splenic dendritic cells were isolated as previously described (Wi tner-Pack et al., J.Exp.Med,166:1484 (1987)), supplemented with rGM-CSF (200U / ml) Cultured from low density spleen adherent cells in medium with or without keratinized or keratinocyte conditioned medium.   Using a two-color labeling method, a specific subset of leukocytes (PE-labeled antibody) and DEC-205 (NLDC-145 rat mAb or rabbit polyclonal anti-DEC-205 followed by FITC labeling Anti-Ig) were identified at the same time. The non-reactive control antibody was non-immune rat IgG2a (dimme Do, Inc., South San Francisco, CA) and rabbit IgG (Jackson Immunolisa) H: Complete IgG or F (ab ')_TwoWas prepared by us). The order of staining is: (a) Primary (B) secondary anti-Ig (mouse anti-rat IgG or goat anti-c Egret F (ab ')_TwoAnd FITC complex, both obtained from Jackson Immunoresearch) c) for quenching, rat or rabbit IgG 10 μg / ml; and (d) PE- or biotin -A labeled antibody. The last reagent was from Farmingen (La Jolla, CA) Purchased: Biotin complex is class II MHC (clone AMS-32.1), B220 / CD45RB ( Clone RA3-6B2) and Thy-1.2 / CD90 (clone 53-2.1) antigen; or PE The complex was composed of Mac-1 / CD11b (clone M1 / 70) and Gr-1 granulocytes (clone RB6-8C5) Determined as antigen. At least 10,000 cells per sample should be used for FACScan fluorescent cell counts. Analyzer (Becton Dickinson Immunocytometry Systems, Inc. New York, CA).   Immunoblotting-This includes, as mentioned above, monoclonal and polyclonal The reaction was carried out with the internal reagent (Example 1 described above).   Immunocytochemistry-Cytospine using pure acetone at room temperature Fix for 10 minutes, air-dry, and stain with antibodies exactly as in fluorescent cell analysis. Was. Same secondary except that peroxidase conjugate was used instead of FITC conjugate Reagents were used. Staining was visualized with diaminobenzidine (Stable DAB, Lisa -Genetic, Hansville AL).   Functional testMonoclonal (10 and 1 μg / ml) and polyclonal (30 and 10 μg / ml) antibody at two doses at near-saturated or above-saturated doses. 3 × 10^FiveOf lymph nodes that passed through nylon wool T cells were implanted with graded doses of allogeneic X-irradiated or mitomycin-treated DC. (Inaba and Steinman's paper, J. Exp. Med.,160:1717 (1984); Inaba et al. Paper, J, Exp.Med,166:182 (1987)). Syngeneic MLR was performed almost simultaneously. ML Positive controls for R inhibition include reagents that interfere with the B7 costimulatory system (GL against B7-2). -1 rat mAb) (Hathcock et al., Science,262:905 (1993)). In in vivo experiments, we performed a local graft-versus-host (GVH) reaction (Atkin and d thesis, J.Exp.Med.,141:P. 664 (1975)), where the parental Lymph node cells induce GVH in the draining popliteal node of F1 mice, presumably F1 dendritic cells appeared in the lymph node cortex. Control (PBS injection) lymph nodes and GVH nodes Was weighed on day 7 using 5 mice per group. This protocol Inject 200 μg of anti-DEC-205 or control IgG into the footpad at time 0 and Is repeated every 8 hours thereafter.⁷Parent lymph node cells or corresponding amount of PBS Was added.result   Expression of DEC-205 by epithelial dendritic cells-3 antibodies against DEC-205 (monoclonal Lonal NLDC-145, polyclonal anti-DEC-205 IgG, and polyclonal anti-DEC-2 05 F (ab ')_TwoFragment) was allowed to act on the cultured epithelial cells. The data so far is NL Intense staining of Langerhans cells with DC-145 was shown. The antigen of this NLDC-145 The determinant is trypsin sensitive (below) and using trypsin first, We prepared an epidermal sheet from which Langerhans cells were released, so we cultured it overnight. The epithelial cells thus obtained were concentrated to increase the amount of the protein on the cell surface. This culture During the fermentation period, most keratinocytes adhere to the plastic surface and are non-adherent We also provided time for Ngelhans cells to achieve low flotation densities (Crowley et al., Supra). Schuler and Steinman, supra). As a result, 20-50% dendritic cells Of the preparations in a non-adherent, overnight culture test of epithelial cells, especially on dense BSA columns. Could be obtained after ascent.   Epithelial cells were phycoerythrin (PE) labeled for class II MHC proteins Stain with mAb to differentiate Langerhans cells from keratinocytes and to differentiate into other leukocytes In contrast, the cells were counterstained with NLDC-145 and hybridoma supernatants of mAbs (FIGS. 13A-D). The specificity of NLDC-145 for dendritic cells (FIG. 13A) was determined by macrophages (SER-4 anti-sia Loadhesin (Coocker and Gordon, J. Exp. Med.,169:1333 (1989)), B Cells (RA3-6B2 anti-B220 (Hoffman and Weissman, Nature,289:681 pages (198 1))), and T cells (53-6.72 anti-CD8 (Ledbetter and Herzenberg, Immunol. Rev.,47: P. 63 (1979))), isotype-matched IgG2a mAb Revealed the fact that it was not reactive with NLDC-145 (+) cells (Figure 13, panel Le B-D). The same suspension is used for purified NLDC-145 IgG (rat IgG2ak) or non-reacted As a sex control, use a graded dose of any of the polyclonal non-immune rat IgG2a In the case of counterstaining, staining of Langerhans cells with NLDC-145 was performed at 2 μg / ml. (Figure 13, comparison of staining of MHC-II (+) dendritic cells with arrows in panels E-G, H) .   Furthermore, this F (ab ')_TwoAnti-DEC-205 rabbit polycloth, both fragmented and whole IgG Non-immune F (ab ') for a wide range of doses (0.3-100 μg / ml)_TwoPassing And IgG. This rabbit reagent reacts with class II MHC-negative keratinocytes But the staining was comparable for immunological and non-immune reagents, This binding was completely non-specific (FIG. 13, panels I-L and M-P, and Q-T and U-X comparison). However, staining of MHC-II (+) Langerhans cells was dark, specific and , And complete IgG or F (ab ')_TwoFragment anti-DEC-205 antibody at an apparent saturation of 30 μg / ml (Fig. 13, F (ab ')_TwoAbout panel I-L and IgG Panel Q-T).   The second rabbit polyclonal antibody is a synthetic antibody that spans the first 19 residues of DEC-205. Occurs in the peptide, which does not stain Langerhans cells and instead replaces non-immune IgG-like (Not shown).   The DEC-205 antigenic determinant was tested for trypsin sensitivity. Cultured LA partially concentrated Treated cells with trypsin (0.25%, PBS solvent, 30 minutes on ice) or One of the exposures was performed. For both monoclonal and polyclonal reagents When stained, it was 10 times lower (A and B in FIG. 14 were compared with C and D, 1 log). During subsequent overnight cultures, these antigenic determinants are re-expressed and untreated. (E and F in FIG. 14 were compared with A and B in FIG. 14).   Expression of DEC-205 by other dendritic cell populations-Splenic DCs with a low buoyant density splenocyte suspension In (see Example 1: and Surggard, supra) their integrin CD11c (FIGS. 15A, C.E and G) were detected with mAb N418 (Metlay et al., J.E. xp.Med,171:1753 (1990)). NLDC-145 or anti-DEC-205 polyclonal Counterstaining with any of the antibodies can be performed on these fresh, isolated or overnight cultured The cells expressed less DEC-205 on their surface than Langerhans cells (FIGS. 15C, D, G and H). Fresh and isolated splenic DCs are It contained two phenotypic subtypes (Crowley et al., Supra). Almost (ho About 80%) express relatively low but detectable levels (≦ 1 log fold) of antigen, The smaller group stained moderately (ca. 1.5 log-fold: arrow, FIGS. 15C, D, G and H). one After overnight culture, the expression of DEC-205 by all CD11c (+) DCs was moderate (1.5 log Level), but never the level observed in epithelial dendritic cells (ca.2 log fold: arrow Mark, Fig. 15K, not L, O and P), but also by cell culture in rGM-CFS. Or enhanced expression of DEC-205 in conditioned medium of keratinocytes containing GM-CFS (upr egulate), but the yield did not increase beyond the amount induced by culture alone (Fig. Not shown).   In contrast, when dendritic cells were derived from bone marrow progenitor cells by GM-CSF (see Ind, supra). aba et al. (1972) reported that their DEC-205 expression was consistently high and that It was equivalent to the level of Hans cells (not shown). Interestingly, bone marrow cultures The day 6 active growth group contained relatively few cells that expressed DEC-205. However, expression was consistently high up to day 8 (FIG. 16).   Expression by resident and induced peritoneal cells-Resident peritoneal cells (about 30 macrophages % And B cells 70%), concanavalin A (ConA), thioglycolate (TGC) Or in exudates induced by the viable microorganism M. bovis calmette gellan (BCG) Comparison with inflammatory peritoneal cells (FIG. 17). This data shows that polyclonal non-immune And immune F (ab ')_TwoFragments are shown. Same for NLDC-145 monoclonal (Not shown). However, non-immune and immune complete rabbit IgG Produced strong background staining in peritoneal macrophages, which was probably due to Fc Probably due to binding to the receptor (not shown). Resident Mac-1 (+) peritoneal macro The phage did not express surface DEC-205, but the peritoneal B cells had measurable levels. (Approximately 1 log times or more than the background: arrows in FIG. 17E-H). Macrophages in ConA and BCG-induced exudates were also reduced by anti-DEC-205. Or they did not stain at all (Fig. 17E-H, arrowhead), but these macrophages All were strongly class II MHC-positive (not shown). This ConA and BCG exudation liquid Contained a significant number of T cells, which did not stain with anti-DEC-205 (Anti-Thy-I double label, not shown). TGC-induced, in contrast to the other abdominal groups tested Macrophages produce low levels of DEC-205 (0.5 to 1 log times background). ). B cells stained equally in each of the resident and provocative groups.   Expression of DEC-205 by resident leukocytes of multiple tissues, especially B cells-Resident abdominal cavity Given the surprising finding that B cells express DEC-205, we Transfer cell suspensions from spleen, bone marrow, peripheral blood, lymph nodes and thymus to several leukocyte And tested for DEC-205 co-expression in Examined. The results for the first three organs are shown here (Figure 18). About spleen and lymph nodes The results were the same (not shown). Thymocytes only, above background It stained very slightly (<0.5 log times) strongly (not shown).   In the spleen suspension, B cells (B220 and MHC-II (+); Gr-I, Mac-1 and Thy-1 (- ): Figures F-J, arrows) also show DEC-205 expression and approximately one pair of background. Stained several times or more. T cells (Thy-1 (+)) and granulocytes (Gr-I (+), Mac-1 (+)) , Also stained, but thinner than B cells.   In bone marrow (Fig. 18K-T), granulocytes were almost one log times background. In contrast, bone marrow B cells exhibited heterogeneous levels of DEC-205. Ho Most B220 (+) cells stain weakly or not at all in the bone marrow However, higher levels of B220 cell subsets (Figure 18P-T, arrow, Co-expressed DEC-205 at the same level as peripheral blood B cells. These B220^{br ight} DEC-205 (+) cells also express surface IgM and these have been identified as mature B cells ( Not shown). A small subset of Mac-1 (+) and B220 (-) cells, probably monocytes. Did not express DEC-205.   In peripheral blood (Figure 18U-δ), B cells (arrows) are identical to B cells in lymphoid tissue. However, granulocytes and T cells showed weak but measurable staining.   To gain insight into the relative amount of DEC-205 expressed in different cell types (Figure 19) , Bone marrow dendritic cells, splenocytes (about 65% B cells) and peripheral blood (about 70% B cells and 30% Lysate of NP-40 from whole cells of E. did. The signal of 10,000 BMDCs is about twice as high as the signal of 100,000 splenocytes strength Power, which means that DEC-205 per cell in the BMDC can reduce the number of splenic B cells Also corresponded to 10 times. The signal of 100,000 peritoneal cells is even weaker, 5-10 times weaker than the signal at 10,000 DCs, which is due to DEC-205 in peritoneal B cells. However, it corresponded to about 50 times less than BMDC.   To test the effect of B cell activators on DEC-205 surface levels, B cells were grown for 2 days in the presence of LPS, CD40 ligand, and a combination of anti-IgM and IL-4. The culture was continued for a while. The surface level of DEC-205 is monoclonal and polyclonal No significant increase was elicited when detected with both reagents, but the last combination Induced a moderate (2-fold) decrease (not shown).   The viability of mouse B cells is greatly enhanced during stimulation in culture, We found that the DEC-205 antigenic determinant on B cells was adsorbed to its surface from extracellular sources Rather than being actively synthesized by this cell. . For B cells, as in Langerhans cells, trypsinization was performed with NLDC-145 and NLDC-145. Most of the staining with anti-DEC-205 was removed, but its antigenic determinants remained Was synthesized again.   Attempts to block dendritic cell function with antibodies to DEC-205-Unidirectional mixed phosphorus The papilla reaction was performed using 10 μg / ml NLDC-145 or 30 μg / ml polyclonal anti-DEC-205 antibody. No inhibition of allogeneic T cell proliferation when performed in the presence of either (FIG. 20). Costimulatory protein B7-2 (Hathcock et al., Supra; Freeman et al. Dissertation, J.Exp.Med.,178:2185 (1993); Inaba et al., J. Exp. Med.,180:1849 Rat IgG2a monoclonal GL-1 (p. (1994)) as a positive block control. Used. GL-1 inhibited the growth of this system, but did not terminate it. But from this: Blocking multiple costimulators completely eliminates allogeneic MLR growth. Is necessary to remove them (Young et al., Clin. Invest.,90: 229 pages (1992)) Was expected.   Dendritic cells express DEC-205 at high levels (see Kraal et al., Supra; Example 4) In homing T cells to T-dependent regions of lymphoid tissue, DEC-205 Local graft-versus-host (GVH) reactions to determine if (Atkins and Ford, supra). Inject parent node lymph node cells into hind footpad When fired, a reaction occurs in the drained popliteal lymph nodes of F1 mice. T thin The vesicle migrates to the draining node, where it is probably first on dendritic cells and Within homologous T cell regions, allogeneic MHC proteins are encountered. Non-immune or anti-DEC-205 F ( ab ')_TwoA substantial dose of the fragment (200 μg) was injected at time 0 into the footpad. 8 hours later, twice An eye antibody dose was injected along with 10,000,000 lymph node cells (ca. 70% T cells). Was. These T cells trigger a strong GVH response: draining lymph nodes Inside, it swelled to 5 to 6 times the normal size. Polyclonal anti-DEC-205, in vi vo failed to inhibit this primary T cell response (Table 3). Consideration   Previous studies have shown that most types of leukocytes other than dendritic cells have DEC-205 antigen. The detection failed (Kraal et al., Supra; Crowley et al., Supra). Real truth In the examples, low levels of other leukocytes, especially B cells, express DEC-205. It became clear that it could come. Example 4 demonstrates that DEC- We report 205 low-level expression. Poly to the N-terminal peptide of DEC-205 Clonal rabbit antibodies and the complete protein identified in Example 1 were used. Was used. This anti-peptide reagent cannot stain viable cells, which Indicates that, under natural conditions, the amino terminus of DEC-205 relates to a 19-residue synthetic immunogen. Change its conformation or steric hindrance of access by antigens , Suggesting that it is related to higher order protein structures. Anti pepti The failure of dopolyclonal to bind to cells indicates that the tissue N-terminal Depending on the covalent modification of phosphorus, this residue is present in proteins isolated from the thymus. And is susceptible to Edman degradation, so it cannot be explained. By contrast, complete The polyclonal against the new DEC-205 has a pattern of reactivity very similar to NLDC-145. Cells associated with the cells were stained.   Differences in DEC-205 expression between different classes of leukocytes were observed. In addition to dendritic cells B cells expressed the largest DEC-205, but their levels were 10-50 It was twice lower (Fig. 19). After trypsinization, the DEC-205 antigenic determinant is As it is regenerated, DEC-205 detected on the surface of B cells actively integrates with the cells themselves. It is not expected to be adsorbed on its surface from extracellular sources Was. DEC-205 expression is expressed in bone marrow from pre-B cells to surface IgM (+) B cells. Obviously, it is equivalent to metastasizing lively. But various mitoji Stimulation of peripheral B cells by the enzyme (LPS, CD40 ligand, anti-IgM + IL-4)  Was not accompanied by a significant increase in the surface expression of. Granulocytes contain DEC-205 more than blood It was expressed at higher levels in bone marrow granulocytes. Thymocytes and spleen and lymph Nodal mature T cells express very low but detectable levels of DEC-205 while T cells in blood and ascites did not express detectable levels. This granulocyte And DEC-205 detected on T cells are probably in bone marrow stroma, thymic epithelium, and peripheral Surrounding stromal cells rich in DEC-205, such as dendritic cells in the T cell area of lymphoid tissue Will be adsorbed from As mentioned earlier (Wiffels et al., Immunobiol.,18 Four: P. 83 (1991)) that cells induced with thioglycolate are weakly positive. Nevertheless, most macrophage populations do not have DEC-205.   Other leukocyte lineages also express DEC-205, but have been evaluated by immunoblotting. Thus, it is clear that dendritic cells express the antibody 10 to 50 times more. DEC- The expression of 205 is regulated on dendritic cells in several ways. Fresh isolated Splenic DCs have relatively low DEC-205 and their levels are It only increases crab. Dendritic cells expressing high levels of DEC-205 are Dendritic cells in the T cell region of peripheral lymphoid organs and proliferating in the presence of high dose GM-CSF Dendritic cells grown from bone marrow progenitor cells.   To examine the contribution of DEC-205 to the immune response in tissue culture systems, we Attempted to inhibit primary allogeneic MLR. DEC-205 interacts with helper T cells It was postulated to be important in the ability of the dendritic cells to work. However, things Both clonal and polyclonal antibodies are available in monoclonal or polyclonal forms of this T cell. Could not block the reaction with any of the primary antibodies. Furthermore we are in vivo Try to block the local GVH response, which in this case Injected parental T cells drain this injection and bind to F1 dendritic cells and B cells. Then migrated to lymph nodes that had begun alloreactive reactions. Similarly, DEC-205 Antibodies did not show any effect. For these negative results, General explanations, such as previous or compensatory resynthesis of new DEC-205 proteins. Can be Assuming that the molecular weight of DEC-2055 is high, monoclonal And polyclonal antibodies both interfere with their function in the studied reaction. It is possible to bind to an antigenic determinant on a natural protein, such as not present. Ah Alternatively, the assay system is designed for systems where DEC-205 does not play a significant role. Stem. The putative function of DEC-205 in the immune response depends on the events studied here. Even earlier events, such as antigen acquisition by accessory cells, or lymphocytes It is clear that it is related to a selection event in expression and the like. In addition, DEC-205 The function can be examined by the molecular cloning of the antigen described in Example 2 above. It is possible. Example 4: In situ DEC-205 protein in lymphoid and non-lymphoid tissues Expression   In this example, monoclonal and polyclonal antibodies to DEC-205 were Immunohistochemical staining of frozen sections of various organs, and The distribution of DEC-205 was re-evaluated by the no blotting method.   To better understand the tissue distribution of DEC-205, we Both polyclonal antibodies are more sensitive than those used in previous studies Various tissues can be tested histologically and by immunoblot with secondary anti-Ig reagents. (Kraal et al., J. Exp. Med.,163:981 (1986); Crowley et al. Cell Immunol. ,118:108 (1989); Vremec et al., J. Exp. Med.,176:Page 47 (1 992); Austyn et al., J. Immunology,152:2401 (1994); Lu et al., J. Exp. Med.,179:1823 (1994); Breel et al., Immunology,63: 657 (1988)). DE Abundant expression of C-205 is found in thymic and intestinal epithelium and in T lymphocytes in peripheral lymphoid organs. Histologically confirmed in dendritic cells in the cell area. In addition, DEC-205 Some other areas: B cell follicle B lymphocytes, bone marrow stroma, pulmonary airway epithelium, and brain hair It was also visualized in small blood vessels. The immunoblotting method is used for lymphoid tissues, DEC-205 protein is substantially present in lysates prepared from lung, bone marrow, and intestine. Confirmed that it exists. Therefore, DEC-205 is highly expressed by dendritic cells. While expressed, it was also expressed in situ by many other cell types.Materials and methods   mouseThree strains of mature (6-12 week old) female mice were tested: inbred C57B L / 6 x DBA / 2 (Told Institute, Saranac Lake, NY), and B ALB / C and outbred CD-1 Swiss-Webster strains (the latter two strains Kufarm, Germantown, NY).   Immunohistological method-Immediately after removal of the organs, they are treated at -20 ° C with O.C.T. Mills, Elkart, IN) and stored at -20 ° C. Normal thickness 10μ m tissue sections from the Minotome cryostat (IEC Slides with 10 wells (Carlson site) (Entific, Peoton, IL). Use these sections in pure Fixed at room temperature for 10 minutes and air dried. The next step is performed in a humidification chamber. Was. Sections were rehydrated in PBS drops (30-50 μl) and then the primary antibody was applied. Not yet Hybridoma supernatant, either diluted or diluted 1: 5 in PBS + 1% BSA Was used according to its titer. Purified IgGs, ascites fluid and antiserum should be Diluted with the same solvent to obtain the optimal dose determined by titration. Is usually 1-10 μg / ml for purified protein, ascites and hyperimmune serum It was 1: 3000 to 1: 1000. These primary antibodies are NLDC-145mAb, Either hybridoma culture supernatant or purified IgG (protein G eluate) Or using rabbit polyclonal anti-DEC-205 (Example 1 above) IgG (protein A eluate) or F (ab ')_TwoUsed as Positive control is other white blood Sphere subset (RA3-6B2 anti-B220 / CD45RB (TIB 146, ATCC, Rockville, MD), SE R-4 anti-macrophage, 53-6.72 anti-CD-8 (TIB 105, ATCC) and MHC class I Rat mAbs against the I protein (M5 / 114 (TIB 120, ATCC)), plus B Polyclonal antibody against rabbit Igβ, one of the signal chains linked to cell surface Ig (Sanchez et al., J. Exp. Med,178:1049 (1993)). Negative pair Teru is a polyclonal rat IgG2a (Jimed, South San Francisco) , CA) and rabbit IgG (Jackson Immunoresearch, West Grove, PA) ), Complete or F (ab ')_TwoWas one of the fragments. Primary antibody is included in the section Contact for 45 minutes at room temperature, after which the sections were washed 5 times with PBS. These sections did not dry for more than a few seconds. Next, an anti-Ig secondary antibody was added, This is usually the donkey's F (ab ')_Two-Horseradish peroxidase complex (ja (Xson Immuno Research) was added at a dilution of 1: 300 in PBS + 1% BSA. After incubation for 45 minutes, the sections were washed 5 times with PBS. The dye substrate is H_TwoO_Two Preparation containing diaminobenzidine (Stable DAB, Research Genetic, Huntsville, AL). The sections are washed five times with PBS and usually Counterstained with Gill's Hematoxylin # 1 (Fisher, Fairlawn, NJ) . Place the coverslip using Permaunt histological mounting substrate (Fisher). Was.   Extraction and immunoblotting of proteins from multiple organs-Organ is 10 times Amounts of phenol and guanidium isothiocyanate (TRIzol, Gibco-BRL, Gaithersburg, MD) in a one-phase solution (Chomczynski, BioTechniques ,Fifteen: 532 (1993)). Organs were homogenized for 15-30 seconds (Polytron, Brine (Cuman, Westbury, NY). RNA and DNA_ThreeRemoved by extraction, and D Precipitated with ethanol. Protein was isolated from the phenol-ethanol supernatant Precipitate with lopanol (150% of the original TRIzol volume) and 1% SDS (the original TRIzol (30% of 1 volume, 50 ° C., 1 hour). Clarify the extract (3000 xg, 1 0 minutes at room temperature) and the total protein concentration was measured (BCA assay, Pierce, Rockford, IL). Immunoblotting is performed as described above (Chomcz, supra). ynski's paper) and 50 µg of the normalized protein was run per column. fill NLDC-145 IgG 10 μg / ml or 1: 1 diluted mAb 1D4B (anti-LAMP-1) Stained with any of the lidoma supernatants.result   For each organ tested, the NL generated against purified DEC-205 protein Similar conclusions were made for the DC-145 monoclonal antibody and the rabbit polyclonal antibody. The fruit was obtained. It has been described in some lymphoid and non-lymphoid tissues. Will be. In many cases, DEC-205 was found to be expressed in situ. This has not been clarified in previous studies.   Thymus: The staining pattern of this organ was originally shown for NLDC-145 mAb (Kraal et al., Supra). Very powerful peroxidase immunity Labeling was observed in the thymic cortical epithelium, while weak staining was observed in (M, FIG. 21, panels a-c and g).   Lymph nodes: Strong DEC-205 expression is apparent in dendritic form throughout the T cell area of the cortex. (T, FIG. 21, d-f). At high power, plaques stain T-cell areas of the deep cortex Point features were observed (FIG. 21h). This speckled pattern shows the dendritic cells The presence of DEC-205 in the inner granules and / or DEC-205 on the surface of many fine protrusions Is shown. It was clear that the medulla was not stained (M, FIGS. 21d-f). DE Weak staining of C-205 was evident on follicular B cells (B, FIGS. 21d-f). This dye The color was unclear when the nuclei were counterstained using hematoxylin (Fig. 21d-f) was clearly visualized when hematoxylin was not used (FIG. 21i). .   spleen: Strong staining of DEC-205 in the T cell area, (Figure 22: The central artery in the T region is indicated by an arrow in each micrograph. ). The extent of DEC-205 staining in the T region is similar to that seen with anti-MHC class II (FIG. 21, b is compared with c and d and e are compared with f). Actually had spots, as in the lymph node T cell area (described above) (Fig. 22d-e) . Like the lymph nodes (above), B cell follicles (B, Fig. 22a-c) are stained with dec-205. However, the staining was so weak that counterstaining of the specimen with hematoxylin (Figure 22, intense B cell staining, panel a) anti-Igβ and panel c anti-MHC-II compared to panel b anti-DEC-205 staining) . Anti-DEC-205 did not stain marginal areas (due to strong B cell staining, (See arrowhead of marginal sinus, prominent in panels a and c).   brain: Both NLDC-145 and polyclonal anti-DEC-205 reagents And linear along the capillaries (arrows, FIG. 23a-c) and small arteries (arrows, FIG. 23d). Staining occurred. No capillary staining found in any other study Was.   lung: Many intensely stained DEC-205 shapes were scattered in lung parenchyma (Fig. 23e, g, h) . We determine whether these shapes represent dendritic cells, macrophages, or both. It has not been decided yet. Probably alveolar macrophages Some strongly stained cells in the respiratory tract were evident (*, FIG. 23h) . DEC-205 was present in the epithelium of all small airways (arrows, FIGS. 23e, h). In contrast, anti-MH C class II did not stain the airway epithelium but stained cells around the airway (FIG. 23f, Arrow).   Bone marrow: If the bone marrow protrudes from the thigh as a complete plug and is cut The lace pattern of DEC-205 is clearly visible throughout this plug. It is probably a stromal cell in the bone marrow (arrow, FIG. 23i). Dark staining of round cells Most show background staining of eosinophils, which is Is expressed. It is clear that any antibodies are missing (not shown) ).   Upper gastrointestinal tract: Tongue oral epithelium served as an example of stratified squamous epithelium. I Some positive forms of DEC-205 are probably Langerhans cells and Suprabasally (arrows, Figure 23j). Anti-MHC class II antibodies Their intraepithelial dendritic cells stain more frequently and / or more intensely and Many subepithelial tissues above the dermis were stained (not shown).   Lower gastrointestinal tract: Strong staining was observed in the small intestine and large intestine columnar epithelium. This stain Was stronger at the tip of the parenchyma than at the center of the epithelium (FIG. 23k-l). Best cut In a piece, staining is more intense along the basal plane than at the tip of individual epithelial cells Met. Many cells in the lamina propria of the soft protrusions stained dark, and this staining also It is due to endogenous peroxidase of eosinophils.   liver: Staining of DEC-205 is not clear, except for a rare case in portal triad (Not shown).   heart: Staining of DEC205 was not clear (not shown).   kidney: Strong expression of DEC-205 was not observed, but some very weak DEC-205 Staining was scattered on the cortical tubing (not shown).   Distribution of DEC-205 by immuno-blotting of multiple organs:DEC-205 Tampa To examine the tissue distribution of proteins, TRIzol protein extraction from several different organs The product (method section) was analyzed using monoclonal NLDC-145 IgG (FIG. 24A) and polyclonal Immunoblot with both anti-DEC-205 IgG (not shown). Viscera flowed down each row The amount of protein in the lysate should be adjusted to the protein loading (50 μg / row). Standardized. Lysosomal membrane marker LAMP-1 (Chen et al., Arch. Biochem. .Biophys.,239:574 (1985)) shows that an equal number of lysosomes appeared in each row. (FIG. 24B). Among lymphoid tissues, the signal of DEC-205 is Maximal in thymus, and bone marrow and lymph nodes were stronger than spleen . These findings were closely related to the histological staining levels described above. Non-lymphatic In systemic tissues, a strong signal equivalent to that of the thymus is apparent in the lung and intestine. (FIG. 24A). Liver, kidney and brain extracts contained trace amounts of DEC-205.Consideration   Donkey F (ab ') modified to enhance sensitivity of antigen detection_TwoSecondary anti-rat Ig test Using drugs, we have discovered that the NLDC-145 monoclonal antibody Showed that it reacted with more tissue than it had. In particular, clear staining In capillaries, bone marrow stroma, epithelium of intestinal parenchyma and lung airways, and all peripheral B cells were found in the follicles of the lymphoid tissue. These newly recognized DEC-205 Accumulation of antigen is due to tissue, which was initially noted to express DEC-205 protein, Dendritic cells in the T cell area of the thymic cortical epithelium and peripheral lymphoid organs stain more strongly I didn't get it.   The tissue distribution of DEC-205 protein was determined by polyclonal purification against purified DEC-205. Proven by antibodies. Rabbit antibody F (ab ')_TwoAnd full IgG form Resulted in a staining pattern similar to that obtained with monoclonal NLDC-145. Further, this tissue distribution was determined by immunoblotting of monoclonal and polyclonal reagents. Multiple organs, tracking by immunoassay and corresponding to the relative intensity of immunohistological staining The relative concentration of expression in the tissue sections was observed. The exception is the DEC-205 A trace amount of protein can cause hepatic, cardiac, and other deficiencies in identifying depletion of DEC-205 positive cells. This was evident in extracts of organs such as renal kidney. This is very small Of this protein may be present in many cell types, Suggest that it is very low for histological visualization. Example: Sequencing of human DEC-205   The human dec cDNA is a 300 base pair, derived from the 3 'coding sequence of the mouse dec cDNA. Was cloned using the above probe. This human cDNA is used for B-cell lymphoma. High stringency hybridization conditions (0.1 SSC 65 ° C) from library Obtained using The sequence of the human DEC-205 gene was determined and is shown in SEQ ID NO: 7. Estimation The amino acid sequence is shown in SEQ ID NO: 8. The deduced amino acid sequence of SEQ ID NO: 8 is SEQ ID NO: 17. Includes putative fragments after the stop after amino acid 43; There is no and can be ignored.   Human deduced amino acid and nucleic acid sequences are compared to their mouse homologues and These are shown in FIGS. 25A and 25B, respectively. This comparison was made between the proteins and the gene (cD High similarity or sequence similarity with respect to the full length of both protein coding regions Indicates identity.   The present invention is limited in scope by the specific embodiments described herein. This is not to say that such an embodiment describes one aspect of the present invention. Is intended to be, but is not the only, and functionally equivalent This is because certain embodiments are within the scope of the present invention. All molecular weights and nucleotides The size of nucleotide base pairs in terms of numbers is approximate and, for illustration purposes, It will also be appreciated that used. In practice, it is described and discussed herein. Various modifications of the invention, in addition to those set forth above, will be apparent from the foregoing description and accompanying drawings. It will be clear to those skilled in the art. Such modifications fall within the scope of the appended claims. It is intended to be whole.   Various references are cited herein, the contents of which are It is incorporated herein by reference.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 45/00 A61K 48/00 48/00 C07K 14/705 C07K 14/705 16/28 16/28 G01N 33/53 D C12N 5 / 10 33/566 G01N 33/53 33/577 B 33/566 C12N 5/00 B 33/577 A61K 37/02 (72) Inventor Nussenzweig Michael Sea United States of America New York 10021 New York East Sixty Third Street 450 Apartment 3E (72) Inventor Swinger William J. 10021 New York York Avenue, New York, USA 1230 (72) Inventor Ean Wimping, 10021 New York York Avenue, New York, 1230 The Rock Blow Within the University [Continued Summary] Tolerance can be imparted when dendritic cells are at rest, or dendritic cells are activated by stimulation, eg, by cytokines or lymphokines, eg, colony stimulating factor (CSF) At the same time, an immune stimulus (ie, vaccination) can be given.

Claims (1)

[Claims] 1. A method for identifying a ligand for DEC, wherein DEC is located on dendritic cells, on the thymus Endogenous, expressed by skin cells, lung epithelial cells, small intestinal epithelial cells and brain capillaries Membrane protein with an apparent molecular weight of 20 by polyacrylamide gel electrophoresis. It has 5 kDa and has 10 lectin domains, one transmembrane domain and One short cytoplasmic tail with a vesicle localization consensus sequence, And a method comprising the following steps:   a) converting a protein containing at least one DEC lectin domain into a candidate ligand Contacting with; and   b) detecting the binding of the candidate ligand to the DEC lectin domain;     Here, detection of binding between the candidate ligand and the DEC lectin domain is performed. , Wherein the candidate ligand is a DEC ligand. 2. 2. The method according to claim 1, wherein said ligand is a saccharide. 3. A protein comprising at least one DEC lectin domain as described above, Expressed by the cell as a membrane protein, and the candidate ligand is labeled; As a result, the binding of the candidate ligand to the DEC lectin domain results in binding of the cell to the The method of claim 1, wherein the method is detected by detecting a connection of knowledge. 4. The protein containing at least one DEC lectin domain is solubilized And the candidate ligand is irreversibly linked to a solid support, such that The binding of a candidate ligand to the DEC lectin domain results in a solid support of the protein. 2. The method according to claim 1, wherein the method is detected by detecting a binding with the compound. 5. The aforementioned protein containing at least one DEC lectin domain is Irreversibly linked to a carrier and the candidate ligand is labeled so that the candidate Binding of the ligand to the DEC lectin domain results in binding of the solid support to the label. The method of claim 1, wherein the method is detected by detection. 6. A protein comprising at least one DEC lectin domain; 2. The method according to claim 1, which is a Kate DEC protein. 7. The protein having at least one DEC lectin domain is a full-length protein. 2. The method according to claim 1, which is a DEC protein. 8. Human DEC-205 is an integral membrane protein expressed by dendritic cells Has an apparent molecular weight of 205 kDa by polyacrylamide gel electrophoresis, and Lectin domains, one transmembrane domain, and consensus localization of coated vesicles Contains one short cytoplasmic tail with sequence, carboxy-terminal sequence, RHRLHLAGFS C having SVRYAQGVNEDEIMLPSFHD (SEQ ID NO: 1) and Binds to heron polyclonal antibody but reacts with monoclonal antibody NLDC-145 No human DEC-205. 9. 9. The human according to claim 8, which has the amino acid sequence shown in SEQ ID NO: 8. DEC-205. Ten. The nucleic acid is characterized by at least 15 base pairs, A purified protein encoding at least a part of human DEC-205 according to claim 8. Nucleic acid. 11． 10. The nucleic acid of claim 9, which encodes a lectin binding domain. 12． The nucleic acid according to claim 10, which is selected from the following group:   a) a nucleic acid having a sequence corresponding to the sequence set forth in SEQ ID NO: 7;   b) an allelic variant of nucleic acid (a);   c) Polypeptide having the amino acid sequence set forth in SEQ ID NO: 8 up to amino acid 1743 A nucleic acid encoding a tide; and   d) Allelic variants of nucleic acid (c). 13. The DEC protein is an integral membrane protein expressed by dendritic cells Has an apparent molecular weight of 205 kDa by polyacrylamide gel electrophoresis, And 10 lectin domains, one transmembrane domain, and It contains one short cytoplasmic tail with a susceptor sequence and contains at least 15 base pairs. A purified nucleic acid encoding at least a portion of a DEC protein, the nucleic acid comprising Nucleic acids. 14. 14. The nucleic acid according to claim 13, which encodes a human DEC protein. 15． 14. The nucleic acid according to claim 13, which encodes a mouse DEC protein. 16． At least one DEC, wherein the nucleic acid is operably linked to an expression control sequence; Any of claims 10 to 15, which is a DNA molecule encoding a lectin domain An expression vector comprising the nucleic acid according to any one of the preceding claims. 17． A recombinant host cell comprising the expression vector according to claim 16. 18． Chinese hamster ovary cells, African green monkey COS cells, Selected from the group consisting of Din derby canine kidney cells and NIH-3T3 fibroblasts. 18. The recombinant host cell according to claim 17, which is a selected mammalian cell. 19. 18. The method of claim 18, wherein said DNA molecule encodes a full length DEC protein. The recombinant host cell according to the above item. 20. 19. The method of claim 18, wherein said DNA molecule encodes a human DEC protein. A recombinant host cell as described. twenty one. Human DEC-205 is an integral membrane protein expressed by dendritic cells, Has an apparent molecular weight of 205 kDa by polyacrylamide gel electrophoresis and 10 Lectin domain, one transmembrane domain, and consensus localization of coated vesicles It has one short cytoplasmic tail with a row and is Binds to the resulting rabbit polyclonal antibody, but binds to monoclonal antibody NLDC-1 An antibody reactive with human DEC-205 protein, which does not react with 45. twenty two. A human DEC-205, characterized by the characteristics selected from the following groups: The antibody of claim 21:   a) having the following carboxy terminal sequence;     RHRLHLAGFSSVRYAQGVNEDEIMLPSFHD (SEQ ID NO: 1);   b) having the amino acid sequence set forth in SEQ ID NO: 8 up to amino acid 1743; And   c) encoded by a DNA molecule having the nucleotide sequence set forth in SEQ ID NO: 7 Being done. twenty three. 23. The antibody according to claim 21 or 22, which is a monoclonal antibody. twenty four. 23. The antibody according to claim 21 or 22, which is a polyclonal antibody. twenty five. Dendritic T cell areas of pulmonary circulation, intestinal circulation, pulmonary airways, small intestinal tract, skin and lymphoid organs DEC, a molecule that targets tissues selected from the group consisting of cells, thymus, and brain DEC-ligands selected from the group consisting of carbohydrates and anti-DEC antibodies A pharmaceutical composition comprising the combined molecule, as well as a pharmaceutically acceptable carrier. 26. The molecule may be an anti-cancer, anti-viral, antibiotic, anti-parasitic, and anti-inflammatory 26. The pharmaceutical composition according to claim 25, wherein the composition is selected from the group consisting of a drug. 27. Dendritic T cell areas of pulmonary circulation, intestinal circulation, pulmonary airways, small intestinal tract, skin and lymphoid organs In the production of molecules targeting tissues selected from the group consisting of cells, thymus, and brain DEC-ligand is selected from the group consisting of DEC-binding carbohydrates and anti-DEC antibodies Use of DEC-ligand selected. 28. Dendritic cells, thymic epithelial cells, lung epithelial cells, small intestinal epithelial cells, and brain capillaries A recombinant vector for gene transfer into cells selected from the group consisting of , A DEC-ligan selected from the group consisting of carbohydrates binding to DEC and anti-DEC antibodies Recombinant vectors, including DNA vectors linked to DNA 29. The DNA vector is a viral vector, a liposome vector and an exposed The recombinant vector according to claim 28, which is selected from the group consisting of DNA vectors. - 30. A DEC-resin selected from the group consisting of carbohydrates and anti-DEC antibodies that bind to DEC Vaccine containing an antigen caused by a pathogen bound to gand and an immunostimulant . 31. The pathogen is selected from the group consisting of a virus, a bacterium, a parasite, and a tumor. 31. The vaccine according to claim 30, wherein the vaccine comprises: 32. The immunostimulant comprises a cytokine, a lymphokine and an adjuvant 31. The vaccine according to claim 30, wherein the vaccine is selected from the group. 33. Wherein the composition is a carbohydrate that binds to DEC under conditions that do not include an immunostimulant; and A self-antigen or antigen bound to a DEC-ligand selected from the group consisting of anti-DEC antibodies. A composition for inducing immunosuppression, comprising a allergen. 34. The autoantigen is myelin basic protein, collagen or a fragment thereof, DN A, nuclear protein, nucleolar protein, mitochondrial protein, and pancreatic β 34. The composition according to claim 33, wherein the composition is selected from the group consisting of cellular proteins.