Patent Application
20060104975
The invention relates to the field of immunology. The invention in particular relates to the role of dendritic cells in immune responses and to pathogens that are able to capitalize thereon.
Dendritic cells (DC) are professional antigen presenting cells (APC) that induce cellular immunity upon pathogen recognition. These cells are therefore important in the defense against many pathogens1;2;3. Immature DC are seeded throughout peripheral tissues to act as sentinels against invading pathogens4. Upon pathogen capture, DC are activated, process pathogens for antigen presentation on Major Histocompatability Complex (MHC) class II molecules, and migrate to the secondary lymphoid organs where they activate naive T cells to initiate adaptive immune responses1;3;4. Depending on the pathogen that is recognized by the DC, differentiation of naive T cells into Th1 cells is triggered by DC in response to intracellular microbes, whereas Th2-mediated responses are generated by DC to eliminate pathogens residing extracellularly3. Thus DC play an important role in both innate and cellular immune responses against tumors antigens as well as pathogens such as viral, bacterial, fungal and parasitic infections1;5. Knowledge about cell-surface receptors on DC that are involved in recognition of pathogens is only starting to emerge, and include Toll-like receptors (TLR)6;7 and C-type lectins8. TLR recognize specific pathogen-derived components, such as lipoproteins, lipopolysaccharides and bacterial DNA, and relay this information through intracellular signaling cascades leading to the production of regulatory cytokines and upregulation of MHC and costimulatory molecules that lead to activation/maturation of DC6. In contrast, C-type lectins recognize pathogen-derived carbohydrate structures and upon binding internalize pathogens for antigen processing and presentation to T cells8-10. In classical calcium-dependent lectins, conserved amino acid residues in the carbohydrate recognition domain (CRD) are involved in calcium binding and sugar specificity11. A growing number of C-type lectins are described to be specifically expressed by DC. For most of these lectins detailed knowledge about pathogen-targets as well as cellular ligands if any, including the identity of the carbohydrate structure they recognize, is lacking8. The DC-specific C-type lectin DC-SIGN (dendritic-cell specific ICAM-3 grabbing nonintegrin, CD209) is involved in binding of the HIV-1 envelope glycoprotein by DC to enhance infection of T cells12, while the mannose receptor (MR) is involved in recognition of mycobacteria and Fungi/Protozoa13, Some C-type lectins like DC-SIGN can interact with carbohydrate-bearing self glycoproteins (ICAM-2 and ICAM-3) to mediate cellular adhesion processes14;15.
In one aspect of the present invention it was found that a C-type lectin can comprise specificity for more than one type of glycoconjugate. This knowledge is useful for a variety of purposes. For instance, lectins are instrumental in mediating pathogen binding and the presentation of antigens thereof in dendritic cells. This binding property of C-type lectins is utilized by a number of pathogens to at least in part facilitate infection of an individual. Knowledge of glycoconjugate specificity of such lectins therefore provides an entrance to the development of medicaments capable of interfering with the capacity of carbohydrates present on pathogens to interact with the lectin and thereby at least in part interfere with the infection or the severity thereof in the exposed individual. In one embodiment the invention therefore provides a method for at least in part inhibiting the binding of a ligand to a C-type lectin or a carbohydrate binding part thereof, comprising contacting said C-type lectin with an isolated and/or recombinant glycoconjugate comprising at least two mannose residues in α1,2 linkage or a glycoconjugate comprising a fucose residue or a glycoconjugate comprising at least one end standing N-acetylglucosamine or a derivative or multimer thereof. Also provided is the use of a glycoconjugate comprising at least two mannose residues in α1,2 linkage or a glycoconjugate comprising a fucose residue or a glycoconjugate comprising at least one end standing N-acetylglucosamine or a derivative or multimer thereof for at least in part inhibiting the binding of a ligand to a C-type lectin or a carbohydrate-binding part thereof. This method may be used for instance to study the exact binding properties of the lectin. The method is also of use in identifying compounds capable of interfering with an ability of said C-type lectin to bind to pathogens. In a preferred embodiment said method further comprises a cell comprising said C-type lectin. Preferably said cell is an antigen presenting cell, more preferably a dendritic cell or macrophage. In the presence of such cell, the effect of such inhibition may be compared to the function of the antigen presenting cell, for instance on its capacity to present antigen and/or the production of lymphokines and/or cytokines. In a preferred embodiment said C-type lectin comprises DC-SIGN, mSIGNR1, L-SIGN and/or DC-SIGNR. Whereas DC-SIGNR and L-SIGN are predominantly expressed on macrophage/endothelial cells, on lymph node and on liver sinusoidal endothelial cells and DC-SIGN is expressed predominantly on dendritic cells. DC-SIGNR and L-SIGN share the remarkable binding and signaling effects that ManLAM has upon binding to dendritic cells via DC-SIGN. However, whereas DC-SIGN further comprises a particular specificity for glycoconjugates comprising a fucose residue or a derivative or multimer thereof, DC-SIGNR and L-SIGN are lacking such particular specificity. In the present invention there is thus a preference for the C-type lectin DC-SIGN.
With the term “glycoconjugate comprising a fucose residue” is meant a glycoconjugate comprising at least one fucose residue that is α1, 3 or α-1,4-lined to the glycoconjugate. The linking moiety preferably consists of N-acetylglucosamine. The linking moiety is preferably coupled to an oligosaccharide or glycan, which in turn may be part of a larger structure (or carrier molecule) comprising subsequent glycoconjugates comprising a fucose residues, or other compounds such as mannose in any configuration. A derivative of said glycoconjugate comprising a fucose residue comprises the same C-type lectin binding activity in kind not necessarily in amount. A derivative may be generated through modification of the fucose residue. Such modifications may be generated in various ways. Preferably, said fucose is a terminal fucose, i.e. linked via only one α1, 3 or α-1,4 linkage to another molecule or fucose. Preferred examples of glycoconjugates comprising a fucose residue are Lewis bloodgroup antigens Lex, Ley, Lea, Leb and/or LDNF. C-type lectin binding parts, derivatives and/or analogous of the lewis bloodgroup antigens are of course also within the scope of the invention. Different modifications have different effects on the lectin binding properties. Sialylation of Lex (yielding sialyl-Lex, a L-, E- and P-selectin ligand) completely abrogates the recognition by DC-SIGN, indicating that DC-SIGN has a carbohydrate specificity that is distinct from that of the selectins that mediate leukocyte rolling. Sulfation reduces the binding affinity of DC-SIGN for Lex, as well as Lea (
With the term “glycoconjugate comprising at least two mannose residues in α1,2 linkage” is meant a glycoconjugate comprising at least two mannose residues wherein said mannose residues are linked to each other via an α1,2 linkage and wherein one of the mannose residues is linked to the glycoconjugate. The linking moiety preferably but not necessarily comprises mannose in α-1,5 linkage and arabinan or β-D-arabinose. The linking moiety is preferably coupled to an oligosaccharide or glycan, which in turn may be part of a larger structure (or carrier molecule) comprising subsequent glycoconjugates comprising at least two mannose residues in α1,2 linkage, or other compounds such as fucose in any configuration. A derivative of said glycoconjugate comprising at least two mannose residues in α1,2 linkage comprises the same C-type lectin binding activity in kind not necessarily in amount. A derivative may be generated through modification of one or both of the mannose residues. Such modifications may be generated in various ways. Preferably, said at least two mannose residues in α1,2 linkage are terminal residues, i.e. linked via only linkage to another molecule or mannose. In a preferred embodiment said glycoconjugate comprising at least two mannose residues in α1,2 linkage, comprises two or three mannose residues linked to each other via respectively one and two α1,2 linkages. A particularly preferred example of a of glycoconjugates comprising at least two mannose residues in α1,2 linkage is ManLAM. ManLAM comprises a mannose-capped lipoarabinomannan, such as derivable from the cell wall component of Mycobacterium. The mannose-capped lipoarabinomannan is preferably linked to an oligosaccharide or glycan, which in turn may be part of a larger structure (or carrier molecule) comprising subsequent mannose-capped lipoarabinomannans or other compounds such as fucose in any configuration. C-type lectin binding parts, derivatives and/or analogous of ManLAM are of course also within the scope of the invention. Different modifications have different effects on the lectin binding properties. Glycoconjugates comprising two or more sets of at least two mannose residues in α1,2 linkage typically have a higher binding affinity than a glycoconjugate comprising only one set of at least two mannose residues in α1,2 linkage.
With the term “glycoconjugate comprising a N-acetylglucosamine residue” is meant a glycoconjugate comprising at least one end-standing N-acetylglucosamine residue that is linked to the glycoconjugate. The N-acetylglucosamine is preferably directly linked to an asparagin of the glycoconjugate. The glycoconjugate can in turn be part of a larger structure (or carrier molecule) comprising subsequent glycoconjugates comprising N-acetylglucosamine residues, or other compounds such as mannose or fucose in any configuration. A derivative of said glycoconjugate comprising a N-acetylglucosamine residue comprises the same C-type lectin binding activity in kind not necessarily in amount. A derivative may be generated through modification of the N-acetylglucosamine residue. Such modifications may be generated in various ways. Preferably, said N-acetylglucosamine is a terminal N-acetylglucosamine, i.e. linked to the asparagin. Preferred examples of glycoconjugates comprising a N-acetylglucosamine residue. A derivative of a glycoconjugate comprising a N-acetylglucosamine residue thus comprises a modification wherein said modification allows binding of said glycoconjugate to said C-type lectin. Glycoconjugates comprising multiple N-acetylglucosamine residues typically have a higher binding affinity than a glycoconjugate comprising a single N-acetylglucosamine residue.
A glycoconjugate can be mono- or polyvalent for the respective mannose, fucose or N-acetylglucosamine residues. A polyvalent glycoconjugate comprises at least two separate antennae wherein at least two said antennae comprise said residues. In a preferred embodiment a polyvalent glycoconjugate comprises at least two antennae having a terminal fucose, i.e. linked via only one α1,3 or α-1,4 linkage to the glycoconjugate or to another fucose residue. In a preferred embodiment a polyvalent glycoconjugate comprises at least two antennae having at least two mannose residues in α1,2 linkage. In yet another embodiment a polyvalent glycoconjugate comprises at least two antennae having a terminal N-acetylglucosamine, i.e. linked via only one linkage to the glycoconjugate. A multimer is preferably a polyvalent glycoconjugate
A binding molecule can be specific for a monovalent or a polyvalent glycoconjugate. Binding of the binding molecule is typically stronger to the polyvalent glycoconjugate.
Binding of the C-type lectin and more in particular, subsequent action of the cell carrying the C-type lectin is typically significantly higher in case of binding of a polyvalent glycoconjugate. This is in particular so for glycoconjugates comprising at least one end standing N-acetylglucosamine residue.
Carrier molecules may further comprise a large variety of different molecules including peptide, protein, lipid, polysaccharide and also synthetic molecules not expressly mentioned here. The carrier molecule(s) may of course be part of an even larger structure such as a biological or artificial surface, a virus, a bacterium or a eukaryotic cell.
Binding of the ligand to the C-type lectin can be inhibited by interfering with binding site on the ligand, with binding site of the C-type lectin or both. Interference with the binding site of the ligand can be done using a ligand binding molecule capable of specifically binding to the C-type lectin binding part on the ligand. This is preferably done with a proteinaceous molecule such as a carbohydrate specific antibody preferably specific for a glycoconjugate comprising at least two mannose residues in α1,2 linkage or a glycoconjugate comprising a fucose residue or a glycoconjugate comprising at least one end standing N-acetylglucosamine or a derivative or multimer thereof. The experimental part describes non-limiting examples of such antibodies. Non-limiting examples of antibodies comprising a specificity for a glycoconjugate comprising a fucose residue or a derivative or multimer thereof are SMLDN1.1 or SMFG4.1, 6H3, Hp151, 4D2, 54.1F6A, NAM61-1A2 or SMLDN1.1. The ligand binding molecule can also be a C-type lectin, preferably a soluble derivative thereof, comprising specificity for a glycoconjugate comprising at least two mannose residues in α1,2 linkage or a glycoconjugate comprising a fucose residue or a glycoconjugate comprising at least one end standing N-acetylglucosamine or a derivative or multimer thereof. A non-limiting example of a soluble C-type lectin of the invention is a soluble DC-SIGN-Fc chimeric molecule comprising amino acid residues 64-404 fused at the C-terminus to a human IgG1-Fc fragment as described for instance in Geijtenbeek et al (Geijtenbeek et al 2002, J. Biol. Chem. 277:11314-11320). However, soluble C-type lectins comprising one or more of the provided specificities can be generated in various ways (for instance by derivation from DC-SIGNR or L-SIGN). Now that the present invention describes one chimeric molecule others can be generated, for instance as described in Fawcett J et al (Fawcett J et al 1992, Nature 360:481-4).
Interference with the binding site on the C-type lectin can of course also be done in various ways. Interference with the binding site on the C-type lectin can be done using a C-type lectin binding molecule capable of specifically binding to the ligand binding part on the C-type lectin. This is preferably done with a proteinaceous molecule such as a C-type lectin specific antibody or with a glycoconjugate comprising at least two mannose residues in α1,2 linkage or a glycoconjugate comprising a fucose residue or a glycoconjugate comprising at least one end standing N-acetylglucosamine or a derivative or multimer thereof, wherein said glycoconjugate or both may be part of a larger structure. Binding can of course further be inhibited by sterically hindering the binding of the ligand to the C-type lectin. For this it is not absolutely required that the interfering molecule itself binds to the association site of the carbohydrate and the C-type lectin. Binding can also be interfered with successfully steric hindrance by binding of the interfering molecule in the vicinity of the site of association.
The ligand may be any type of structure capable of binding to said C-type lectin. Usually this will comprise a proteinaceous molecule or a carbohydrate. The ligand may be an antibody. It is preferred, but not strictly necessary, that the ligand comprises mannosilated glycans such as cell wall component of Mycobacterium ManLAM, or a mannose derivative, lipophosphoglycan such as derived from Leishmania, or a glycoconjugate comprising at least two mannose residues in α1,2 linkage or a glycoconjugate comprising at least one end standing N-acetylglucosamine or a glycoconjugate comprising a fucose residue such as SEA or CD66 or a derivative or multimer thereof The ligand can have instead of, or in addition to, a glycoconjugate comprising at least two mannose residues in α1,2 linkage or a glycoconjugate comprising a fucose or a glycoconjugate comprising at least one end standing N-acetylglucosamine or a derivative or multimer thereof, different glycoconjugates capable of binding to the same on the C-type lectin.
The ligand preferably comprises an antigen. The ligand can, as mentioned above, comprise a glycoconjugate comprising a fucose residue, at least two mannose residues or at least one end standing N-acetylglucosamine. In this embodiment the antigen does or does not comprises said glycoconjugate. In a preferred embodiment the antigen comprises said glycoconjugate. The ligand preferably comprises a (tumor) antigen, a pathogen and/or a cell associated receptor. An antigen is here used to include peptides or glycolipids and derivatives thereof capable of being presented in the context of MHC class I or class II, or CD1b. At least some C-type lectins, and particularly those expressed on dendritic cells are involved in the process of antigen uptake and presentation thereof by antigen presenting cells. An early step in this process is the capture of the antigen by such lectins. With a method of the invention it is possible to at least in part interfere with this initial step in the process of antigen presentation. Considering the importance of antigen presentation in the initiation and maintenance of antigen specific immune responses, it is clear that the method of the invention can be favorably used to at least in part diminish the potency of an immune response against said antigen. In a particularly preferred embodiment the ligand comprises a pathogen or a C-type lectin binding part thereof. It has been found that many different pathogens comprise carbohydrates capable of binding to C-type lectins of the invention. In particular it has been found that pathogens or parts thereof can bind to said C-type lectins through a mannose containing glycoconjugate such as a glycoconjugate comprising at least two mannose residues in α1,2 linkage or a glycoconjugate comprising a fucose or a glycoconjugate comprising at least one end standing N-acetylglucosamine or derivative or multimer thereof This binding at least in part facilitates infection of an individual with said pathogen. Interference with the binding of the pathogen or part thereof to the C-type lectin can thus at least in part aid an individual in combating an infection by the pathogen and in cases even prevent the establishment of a clinically visible symptoms thereof. The latter is useful in (passive or active) vaccination strategies. The pathogen can be a virus, a fungus, a (myco)bacterium and/or a parasite. The pathogen preferably comprises a glycoconjugate comprising at least two mannose residues in α1,2 linkage or a glycoconjugate comprising a fucose residue or a glycoconjugate comprising at least one end standing N-acetylglucosamine or a derivative or multimer thereof Examples of preferred pathogens are human immunodeficiency virus, a helicobacter, a neisseria meningitidis, a leishmania, a schistosoma, a klebsiella, a probiotic lactobacillus, hepatitis C virus, a herpes simplex virus or an ebola virus. Inhibition of binding of the specifically mentioned pathogens can be achieved using a glycoconjugate comprising at least two mannose residues in α1,2 linkage or a glycoconjugate comprising a fucose residue or a glycoconjugate comprising at least one end standing N-acetylglucosamine or a derivative or multimer thereof In a particularly preferred embodiment inhibition of binding is achieved using a ligand (pathogen or part thereof) binding molecule capable of specifically binding to the C-type lectin binding part on the ligand. This is preferably done with a proteinaceous molecule such as a carbohydrate specific antibody preferably specific for a mannose carbohydrate such as a glycoconjugate comprising at least two mannose residues in α1,2 linkage or a glycoconjugate comprising a fucose residue or a glycoconjugate comprising at least one end standing N-acetylglucosamine or a derivative or multimer thereof The experimental part describes non-limiting examples of such antibodies. The ligand binding molecule can also be a C-type lectin, preferably a soluble derivative thereof, comprising specificity for a mannose carbohydrate such as a glycoconjugate comprising at least two mannose residues in α1,2 linkage or a glycoconjugate comprising a fucose residue or a glycoconjugate comprising at least one end standing N-acetylglucosamine or a derivative or multimer thereof A non-limiting example of a soluble C-type lectin of the invention is a soluble DC-SIGN-Fc as mentioned above. Thus infections of an individual with said pathogens can at least in part be prevented or treated by administering to said individual a mannose carbohydrate such as a glycoconjugate comprising at least two mannose residues in α1,2 linkage or a glycoconjugate comprising a fucose residue or a glycoconjugate comprising at least one end standing N-acetylglucosamine or a derivative or multimer thereof. In a preferred embodiment a ligand binding molecule of the invention is administered to said individual. This embodiment can also be used to at least in part inhibit the binding of the pathogen to a C-type lectin expressing cell thereby at least in part inhibiting the contamination or spread of the pathogen in the body of a patient, as well as inhibiting DC maturation and cell adhesion, as is also described below.
C-type lectins not only recognize carbohydrate profiles on pathogens but also interact with self glycoproteins to mediate cellular processes such as differentation and migration. A method of the invention can therefore also be used to interfere with the interaction with one or more self-glycoproteins and thereby be used to at least in part inhibit the cellular processes that the mentioned C-type lectins are involved in. This can be done by providing the carbohydrates structures provided by the invention (i.e. mannose containing glycojugate such as a glycoconjugate comprising at least two mannose residues in α1,2 linkage or a glycoconjugate comprising a fucose residue or a glycoconjugate comprising at least one end standing N-acetylglucosamine or derivative or multimer thereof). Preferably, this is achieved using a ligand binding molecule of the invention. Thus in another preferred embodiment the method is used to at least in part inhibit binding of said C-type lectin to a self-glycoprotein preferably a receptor present on the outer membrane of a cell. Preferably said receptor comprises ICAM-2, ICAM-3, CD11b, CD166 or CD66 or a functional part, derivative and/or analogue thereof CD166 and CD66 are present on a subset of NK cells and granulocytes. Within the subset of CD66 variants the receptor preferably comprises CD66a and/or CD66e. Thus interaction of these cells with a cell comprising a mentioned C-type lectin can be at least in part inhibited using a specific binding partner for a C-type lectin mentioned above. Thus interaction of preferably dendritic cells and said subset of NK cells and granulocytes can be interfered with. Through this interaction it is possible to modify an immune response. For instance, by at least in part inhibiting binding of a granulocyte to a dendritic cell, through providing a binding molecule of the invention, the C-type lectin of the invention, it is possible to at least in part inhibit the mounting of an effective immune response against antigens carried by the granulocyte. On the other hand it is possible to augment or induce an effective antigen specific immune response by providing the antigen presenting cell with a ligand comprising said antigen and a glycoconjugate comprising at least two mannose residues, a fucose residue and/or an end standing N-acetylglucosamine. Preferably, said C-type lectin comprises DC-SIGN. As specific binding partner of said C-type lectin a mannose containing glycoconjugate such as a glycoconjugate comprising at least two mannose residues in α1,2 linkage or a glycoconjugate comprising a fucose residue or a glycoconjugate comprising at least one end standing N-acetylglucosamine or a derivative or multimer thereof, can be advantageously used. The cellular interaction of DC with granulocytes or NK cells is essential in the innate immune response. In particular granulocytes are involved early ingestion of pathogens and may attract and stimulate DC to participate in pathogen recognition and presentation to T cells. In contrast NK cells are well know to be involved in killing of infected cells. NK cells can kill or activate DC that allow lysis of pathogen captured DC or maturation of pathogen captured DC to enhance T cell stimulation and immune response. The cellular interaction of granulocytes and NK cells with DC is to enhance immunity against pathogens or eliminate pathogen infected DC.
In another aspect the invention provides a method for at least in part inhibiting maturation or activation of an antigen presenting cell comprising providing said antigen presenting cell with a DC-sign binding glycoconjugate comprising at least two mannose residues, a fucose residue and/or an N-acetylglucosamine residue. Using this technique it is possible to for instance dampen an immune response. It is further possible to induce antigen tolerance through this method, one can for instance provide antigen together with the glycoconjugate. In case the antigen present cell is activated through a toll-like receptor pathway the balance between the level of activation/inhibition determines the maturation or activation of the antigen presenting cell. When the TLR pathway is activated strongly whereas the C-type lectin pathway is not, the balance is shifted toward activation of the antigen presenting cell whereas in the reverse situation the balance is shifted toward to tolerazing and inhibition of maturation or activation.
In another aspect the invention provides a method for modulating the stimulating immune effect of a dendritic cell that is preferably activated via a Toll-like receptor signaling pathway, said method comprising contacting said dendritic cell with an isolated and/or recombinant specific C-type lectin binding molecule. Preferably, said C-type lectin comprises DC-SIGN and said specific C-type lectin binding compound comprises a glycoconjugate comprising at least two mannose residues in α1,2 linkage or a glycoconjugate comprising a fucose residue or a glycoconjugate comprising at least one end standing N-acetylglucosamine or a derivative, a combination or multimer thereof and preferably, a glycoconjugate comprising at least two mannose residues in α1,2 linkage, preferably ManLAM. In combination with Toll-like receptor signaling ManLAM binding to DC-SIGN on a dendritic cell induces the production of significant amounts of IL-10 and reduces DC-activation/maturation. The production of IL-10 and reduction of activation/maturation of DC lead to a dampening of immune responses. Thus by providing a glycoconjugate comprising at least two mannose residues in α1,2 linkage or an analogously acting glycoconjugate to a dendritic cell, this cell can be induced to dampen the immune response at least in its direct vicinity. Thus an active immune system comprising a number of dendritic cells participating in the activity can be dampened through by providing the dendritic cell with a glycoconjugate comprising at least two mannose residues in α1,2 linkage or an analogously acting glycoconjugate. This embodiment is useful in situations where an individual is suffering from or at risk of suffering from an over-active immune system. Preferably, said over-active immune system involves graft versus host disease, host versus graft disease and the various auto-immune diseases. Alternatively, an immune system dampened through the discussed mechanism can be stimulated by providing dendritic cells with a specific C-type lectin binding molecule capable of interfering with the binding of a glycoconjugate comprising at least two mannose residues in α1,2 linkage or analogously acting glycoconjugate. ManLAM, at least in amounts active locally, is tolerated by individuals considering that active amounts of ManLAM are secreted by mycobacterium infected cells in the body. Alternatively, the dampened system can be stimulated by using a ligand binding molecule of the invention, preferably a proteinaceous molecule such as a carbohydrate specific antibody preferably specific for any mannose containing glycoconjugate such as ManLAM or a glycoconjugate comprising a fucose residue or a derivative or multimer thereof. Preferably, said antibody comprises a specificity for a mannose residue in α1,2 linkage with another mannose. The experimental part describes non-limiting examples of such antibodies. The ligand binding molecule can also be a C-type lectin, preferably a soluble derivative thereof, comprising specificity for a glycoconjugate comprising at least two mannose residues in α1,2 linkage or a glycoconjugate comprising a fucose residue or a glycoconjugate comprising at least one end standing N-acetylglucosamine or a derivative or multimer thereof Another non-limiting example of such an interfering molecule is AraLAM. This embodiment is useful to combat (chronic) diseases wherein at least part of the phenotype of the disease is due to dampening of the immune system through the mentioned C-type lectin pathway. Preferred examples of such diseases are infections by a human immunodeficiency virus, a helicobacter, a neisseria meningitidis, a leishmania, a schistosoma, a klebsiella, a probiotic lactobacillus, hepatitis C virus, a herpes simplex virus or an ebola virus
Human immunodeficiency virus binds to DC-SIGN through a glycoconjugate having at least two mannose residues. Helicobacter and particularly helicobacter pylori binds to DC-SIGN through a glycoconjugate having a lewisx antigen. Neisseria meningitidis binds to DC-SIGN through a glycoconjugate having N-acetylglucosamine residue. Leishmania mexicana binds to DC-SIGN through a glycoconjugate having at least two mannose residues. Schistosoma mansoni binds to DC-SIGN through a glycoconjugate having a lewis antigen. Klebsiella pneumonea binds to DC-SIGN through a glycoconjugate having at least two mannose residues. Hepatitis C virus binds to DC-SIGN through a glycoconjugate having at least two mannose residues. Herpes simplex virus binds to DC-SIGN through a glycoconjugate having at least two mannose residues. Ebola virus binds to DC-SIGN through a glycoconjugate having at least two mannose residues.
To alter the immune response in a body against the mentioned pathogens, a binding molecule of the invention thus preferably at least in part inhibits the binding of the respective glycoconjugates to the c-type lectin.
Immature DC are highly efficient in antigen capture and processing, whereas mature DC are specialized in the naïve T cell activation necessary for cellular immune responses. Immature DC mature in response to specific ‘danger’ signals such as bacterial components (LPS) or inflammatory cytokines TNFa, PGE2). In the present invention it was found that a glycoconjugate comprising a mannose, a fucose residue or a derivative, a combination or multimer thereof, does not induce DC maturation. In contrast to LPS that triggers TLR4, no up-regulated expression of the activation markers CD80, CD83, CD86 or HLA-DR was observed. However, when the maturation of immature dendritic cells was studied in the situation when the cells are also activated via a Toll-like receptor pathway, a strong inhibition of Toll-like receptor induced maturation was observed, particularly when the dendritic cells were exposed to ManLAM. Such inhibition thus dampens the response of Toll-like receptor pathway stimulated dendritic cells and thereby the capacity of an individual to cope with pathogens that are combated via activation of the Toll-like receptor pathway. This dampening of DC-activation can be at least in part prevented by at least in part inhibiting binding of particularly a glycoconjugate comprising at least two mannose residues in α1,2 linkage, but also analogously acting glycoconjugates to C-type lectins on the immature dendritic cells. The invention thus provides a method for determining whether a compound is capable of modulating an activation state of a dendritic cell comprising providing said dendritic cell with a compound capable of specifically binding to a c-type lectin and determining whether a Toll-like receptor signaling pathway in said dendritic cell is modulated. Further provided is a method for modulating the activity of a Toll-like receptor signaling pathway in a cell, wherein said cell comprises a Toll-like receptor and a C-type lectin, said method comprising contacting said cell with an isolated and/or recombinant C-type lectin binding molecule. Preferably said C-type binding molecule comprises a glycoconjugate comprising a mannose, a fucose residue or a derivative, a combination or multimer thereof. Activity can be modulated upward by at least in part preventing simultaneous stimulation of C-type lectin with a glycoconjugate comprising at least two mannose residues in α1,2 linkage or analogously acting compound. Activity is modulated downward by simultaneously providing said a glycoconjugate comprising at least two mannose residues in α1,2 linkage or analogously acting compound. Analogously acting compounds comprise the same Toll-like receptor signaling interfering capacity as ManLAM in kind not necessarily in amount. A preferred analogously acting compound comprises a glycoconjugate comprising a fucose residue or a derivative or multimer thereof. A further level of control is possible using C-type lectin binding compounds that at least in part interfere with the binding capacity of a glycoconjugate comprising at least two mannose residues in α1,2 linkage or an analogously acting compound. In this way activity of the Toll-like receptor pathway can be modulated upward (in the presence of the proper Toll-like receptor ligand) even in the presence of a glycoconjugate comprising at least two mannose residues in α1,2 linkage or analogously acting compound. The latter feature is of importance in cases where the immune system has problems combating an infection via the Toll-like receptor pathway, particularly when said infection is accompanied with secretion of a glycoconjugate comprising at least two mannose residues in α1,2 linkage or analogously acting compounds, such as mycobacteria and in particular M. tuberculosis and M. Bovis. Thus the invention further provides a method for stimulating maturation of a dendritic cell that is contacted with a Toll-like receptor ligand and a glycoconjugate comprising at least two mannose residues in α1,2 linkage or analogously acting compound, said method comprising providing said dendritic cell with a C-type lectin binding molecule capable of blocking the binding of said glycoconjugate to said C-type lectin. C-type lectin binding molecules capable of at least in part inhibiting the binding of a glycoconjugate comprising at least two mannose residues in α1,2 linkage or analogously acting compounds are for instance C-type lectin binding antibody or a functional part, derivative and/or analogue thereof, with a binding specificity that blocks or covers the ManLAM binding site on said C-type lectin. Preferable said C-type lectin comprises DC-SIGN. Thus in a preferred embodiment said antibody is a DC-SIGN specific antibody. A suitable example of such an antibody is AZN-D1, AZN-D2 or AZN-D3 or a human or humanized analogue comprising the same binding specificity in kind not necessarily in amount. In a preferred embodiment the invention provides a method for stimulating maturation of a dendritic cell that is contacted with a Toll-like receptor ligand and a glycoconjugate comprising at least two mannose residues in α1,2 linkage or analogously acting compound, said method comprising providing said dendritic cell with a ligand binding molecule of the invention, thereby at least in part preventing binding of said a glycoconjugate comprising at least two mannose residues in α1,2 linkage or analogously acting compound to the dendritic cell.
An important aspect of the invention is concerned with the use of a glycoconjugate comprising at least two mannose residues in α1,2 linkage or a glycoconjugate comprising a fucose residue or a glycoconjugate comprising at least one end standing N-acetylglucosamine or a derivative or multimer thereof, a ligand binding molecule of the invention and/or a C-type lectin binding molecule of the invention for the preparation of a medicament. Such medicaments may be used for the treatment of an immune system associated disease or the treatment of an acquired disease. Preferably, said acquired disease comprises an infection with human immunodeficiency virus, a helicobacter, a neisseria meningitidis, a leishmcnia, a schistosoma, a klebsiella, a probiotic lactobacillus, hepatitis C virus, a herpes simplex virus or an ebola virus.
Considering the natural role of C-type lectins on antigen presenting cells it is within the scope of the present invention to stimulate immune responses in an individual by providing antigen through a C-type lectin receptor on the antigen presenting cell. This can of course be achieved when the antigen comprises a a glycoconjugate comprising at least two mannose residues in α1,2 linkage or glycoconjugate comprising a fucose residue or a glycoconjugate comprising at least one end standing N-acetylglucosamine or derivative or multimer thereof In this embodiment it is preferred that simultaneous activation of the Toll-like receptor signaling pathway is at least in part prevented. Provided is thus a glycoconjugate comprising an antigen and a glycoconjugate comprising at least two mannose residues in α1,2 linkage and/or a fucose residue and/or at least one end standing N-acetylglucosamine or a derivative or multimer thereof for use for the preparation of a vaccine. The vaccine may be preventive or curative. The antigen may be derived from any source as long as it is capable of being presented through major histocompatibility complex I, complex II or C1b. In a preferred embodiment said antigen comprises a tumor antigen. The presence of the mentioned carbohydrates on tumor cells facilitates antigen capture by DC to enhance antigen presentation and as a result immune activation. On the other hand, the immune response against tumor antigen lacking the mentioned carbohydrates can stimulated significantly by providing the antigen with one or more of the mentioned carbohydrates thereby stimulating DC uptake and thus the immune response against the antigen. This aspect is useful in the preparation of vaccines.
DC-SIGN has been demonstrated to have a carbohydrate specificity for mannose-containing carbohydrates, Lewis antigens and GlcNAc-containing structures (
DC-SIGN is a C-type lectin in particular highly expressed on Dendritic cells. Its function is to recognize antigen and process and present this very efficiently into MHC class I and II molecules.
It has long been thought that DC-SIGN is a pathogen recognition receptor that recognizes pathogens to activate the immune system, but as it seems now, the long list of pathogens that target DC-SIGN all persist and escape immunity by different mechanisms. We have now four examples of pathogens that target DC-SIGN to survive in the host.
1. HIV-1 and HCV target DC-SIGN to ‘hide’ within DC and escape the intracellular routing to the lysosomal compartment.
2. Secretion of ManLam by mycobacteria targets DC-SIGN to induce downmodulation of TLR induced DC maturation, and induction of IL-10.
3. the human gastric pathogen Helicobacter pylori persists: Lipopolysaccharide phase variants modulate the Th1/Th2 balance through interaction with the dendritic cell lectin D C-SIGN.
4. Lactobacillus target DC-SIGN on DC and induce regulatory T cells, inhibition of recognition of Lactobacillus by DC-SIGN reduces the regulatory state of T cells.
DC-SIGN is thus a receptor that ‘normally’ recognize self-antigen to tolerize (Annual Review Immunology, 2004). We here demonstrate that targeting of DC-SIGN by lactobacilli can indeed induce regulatory T cells. Furthermore it seems that DC-SIGN can also recognize the tumor antigen CEA (CD66e) in particular Lewis X and Y. These tumor antigens can be secreted and suppress DC activation in a similar way as ManLam of mycobacteria tuberculosis. The interaction of DC with granulocytes is elucidated by the fact that DC-SIGN recognizes CD66a and CD11b on granulocytes. In particular Lewis X antigen on these molecules is recognized. In that way DC-SIGN functions as a cell adhesion receptor that mediates cellular interactions of granulocytes with DC. This cellular adhesive function between granulocytes and dendritic cells has never been reported but it is likely that the granulocyte DC interaction is essential to form a bridge between the innate immune response and the adaptive immune response in such a way that the granulocyte passes the infectious agents to the DC mounting an adequate immune response. In particular the granulocyte activates and matures the DC, inducing upregulation of cytokines and costimulatory molecules which is necessary for DC migration and initiation of the adaptive immune response. The interaction of DC with granulocytes is also seen in vivo in the situation of Crohns disease. A new pathogen that interacts with DC-SIGN on DC is Neisseria Meningitidis. Strain variants demonstrate that in particular one strain mutant IgtB interacts which contains endstending GlcNAc residue. Other experiments (
In another embodiment the invention provides the use of ligand binding molecule and/or a c-type lectin binding molecule of the invention for the preparation of a vaccine. A dampening of the immune system due to the effect of a glycoconjugate comprising at least two mannose residues in α1,2 linkage or analogously acting compounds is reduced by providing a glycoconjugate or antibody capable of at least in part inhibiting the binding of a glycoconjugate comprising at least two mannose residues in α1,2 linkage or analogously acting compound to their C-type lectin receptor. The antibody preferably comprises SMLDN1.1, SMFG4.1, 6H3 or SMLDN1.1. Thus antigen present in said vaccine or provided separately is more effective in stimulating or boosting an immune response in the presence of a glycoconjugate comprising at least two mannose residues in α1,2 linkage or analogously acting compound. This is particularly important in patients suffering from a mycobacterial infection, such as but not limited to M. tuberculosis or M. bouis. Preferably, said vaccine is used to stimulate an antigen specific immune response in said individual. In a preferred embodiment the medicament or vaccine is used for the treatment of an individual suffering from a cancer, an autoimmune disease or a transplantation related disease.
As used herein the term “antibody” refers to antibodies derived from humans or other animals. The antibody is preferably produced outside the body. The antibody can also be generated or selected using artificial systems such as phage display selection. An antibody having no natural counterpart is therefore well within the scope of the present invention. Antibodies as used herein also include fragments thereof capable of binding to the same target, such as FAB fragments or even smaller parts. The antigen binding part of an antibody of the invention may also be grafted onto another type of molecule to provide that molecule with a binding specificity as provided for in the invention. Modification of the antibody to include human or humanized versions thereof with the same binding specificity in kind not necessarily in amount are of course also in the scope of the invention. Also included are single chain fragments and variants thereof.
As used herein the term proteinaceous molecule refers to a peptide, a poly-peptide, protein and the like with or without modifications. Such modifications may be synthetic and/or provided for by a biological system. The latter including for instance post-translation modification such as glycosylation.
Where the invention is described for use in humans, the invention is also functional in other animals for instance farm-animals and pets. These medical and vaccine uses and methods of treatment are therefore also part of the invention.
a. Schematic diagram of the covalent structures of the glycan chains of synthetic glycoconjugates used in the DC-SIGN binding assay. b and c. Glycoconjugates were coated and binding of recombinant DC-SIGN-Fc was measured after incubation with peroxidase-labeled goat-anti-human Fc. Anti-DC-SIGN monoclonal antibody AZN-D1 were used to block binding. c. Titration of the glycoconjugates revealed that DC-SIGN binds with high affinity to Lex, Ley, Lea, Leb, or LDNF-glycoconjugates and □1,3,□□1,6 mannotriose, sulfo-Lea, whereas it has a lower binding affinity for □-mannose and □-L-fucose, and sialyl-Lex.
a. Pathogens that consist out of Lex-rich Helicobacter pylori and Schistosoma mansoni, and mannose-capped lipoarabinanomannan (manLAM) of Mycobacterium tuberculosis and mannose-capped lipophosphoglycan of Leishmania mexicana, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus were coated and binding of recombinant DC-SIGN-Fc was measured as with peroxidase-labeled goat-anti-human Fc.
b. DC-SIGN can bind LPS of Klebsiella Len1 that contains mannose cap, but does not bind the LPS of Klebsiella Len 111 that lack a mannose cap indicating that strains that contain the DC-SIGN binding carbohydrates interact with DC-SIGN on Dendritic cells or DC-SIGN transfectants.
a) DC-SIGN interacts with several mycobacteria strains. DC-SIGN-Fc binding to mycobacteria (5*105 bacteria) was determined by a Fc-specific ELISA. Specificity was determined by measuring binding in the presence of blocking DC-SIGN-specific antibodies (AZN-D1 or AZN-D3) and mannan. EGTA was used to determine the calcium-dependency of the DC-SIGN-Fc-mediated binding. ICAM-3-Fc binding to mycobacteria was also measured to exclude non-specific binding by the Fc domain. Standard deviation<0.02 OD450. One representative experiment out of three is shown. Binding of Mycobacterium Smegmatis that lacks the ManLAM structure does not interact with DC-SIGN (data not shown).
b) The schematic structure of ManLAM. Lipoarabinomannan (LAM) of M. tuberculosis consists of a glycosylphosphatidyl anchor (GPI), an mannose-rich oligosaccharide core and a branched arabinose polymer that ends in mannose-caps (n=0-3). AraLAM has a similar structure but does not contain the mannose-cap (ref).
c) The mannosylated-lipoarabinomannan ManLAM, in contrast to the non-mannosylatd AraLAM, is specifically bound by DC-SIGN. The anti-DC-SIGN antibody AZN-D1 was used to determine specificity. The DC-SIGN-Fc binding assay was performed as described in
d) DC-SIGN does not interact with AraLAM. The DC-SIGN-Fc binding assay was performed as described in
a) K562-DC-SIGN transfectants express high levels of DC-SIGN but lack expression of the other reported ManLAM receptors MR, CD11b and CD11c. Transfectants were generated as previously described?. Open histograms represent the isotype controls, and filled histograms indicate the specific antibody staining.
b) DC-SIGN, expressed by K562 transfectants, binds strongly to intact M. bovis BCG and the mycobacterial component ManLAM but not to AraLAM. The adhesion of cells to the LAM glycans was determined using the fluorescent bead adhesion assay. Binding to viable mycobacteria was determined by measuring the binding of K562 transfectants to FITC-conjugated mycobacteria (MOI 20) using flow cytometry. Specificity was determined by measuring binding in the presence of blocking antibodies against DC-SIGN. Standard deviation for the fluorescent bead adhesion assay and the mycobacteria binding assay was <5% and <2%, respectively. One representative experiment out of three is shown.
c) The Val351 amino acid residue is not essential for the interaction of DC-SIGN with M. bovis BCG and ManLAM, similar to HIV-1 gp120, whereas it is essential for ICAM-3 binding. Binding to the V351G DC-SIGN mutant expressed by K562 cells was measured as described in
d)Binding of DC-SIGN to differential components of ManLAM. Neoglycoproteins Ara-Man, Ara-Man2, Ara-Man3 or Ara6 were coated on fluorescent beads and added to DC-SIGN expressing cells. DC-SIGN preferably binds Man2 and Man3 as demonstrated with the strong binding that can be blocked by the addition of anti-DC-SIGN antibodies (AZN-D1).
a) Immature DC express high levels of DC-SIGN and the other reported LAM receptors MR, CD11b and CD11c. Open histograms represent isotype control and filled histograms indicate specific antibody staining.
b) Immature DC bind strongly to ManLAM via DC-SIGN. Binding was determined using the fluorescent bead adhesion assay. Specificity was determined by measuring binding in the presence of mannan, EGTA or blocking antibodies against DC-SIGN (AZN-D2), MR (Clone 19), CD11b (bear-1) or CD11c (SHCL3). Standard deviation<5%. One representative experiment out of three is shown.
c) DC-SIGN mediates capture of M. bovis BCG by immature DC. Binding was determined by flow cytometry using FITC-conjugated mycobacteria. Specificity was determined by measuring binding in the presence of antibodies against DC-SIGN (AZN-D1, AZN-D2 and AZN-D3), MR (Clone 19), CD11b (bear-1) and CD11c (SHCL3). Binding was also measured in the presence of the C-type lectin inhibitors mannan and EGTA, whereas a known MR ligand, mannose-BSA, was used to determine the contribution of the MR receptor. Standard deviation<2%. One representative experiment out of three is shown.
d) DC-SIGN mediates capture and internalization of M. bovis BCG by K562 cells. K562 transfectants were incubated with FITC-conjugated M. bovis BCG (MOI 20). Cells were washed, and surface FITC was quenched by exposure to trypan blue. Phagocytosis was determined by comparing the FITC labeling before and after quenching using flow cytometry. Surface bound bacteria are represented by open bars, internalized by closed bars. Standard deviation<4%. One representative experiment out of three is shown.
e) Immature DC rapidly phagocytose mycobacteria through DC-SIGN. The internalization was determined as described in
a) M. bovis BCG and ManLAM are internalized by DC-SIGN on immature DC and targeted to the lysosomes. The fate of captured mycobacteria was followed by analyzing immature DC pulsed with FITC-conjugated M. bovis BCG (MOI 20) for 2 hours using immuno-fluorescence microscopy (magnification 200×). ManLAM was followed by incubating DC with ManLAM (10 mg/ml) for 1 hour. DC-SIGN, ManLAM and CD207a/Lamp-1 were stained with AZN-D1, F30.5 and H4A3, respectively. One representative experiment out of three is shown.
b) ManLAM induces down-regulation of DC-SIGN, but not of MR, CD11b and CD11c. Immature DC were incubated with 15 mg/ml of ManLAM or AraLAM for 18 hours, and then DC-SIGN expression was determined by flow cytometry. One representative experiment out of three is shown.
a) ManLAM induces IL-10 production of LPS-matured DC. Immature DC were incubated with 15 mg/ml of either ManLAM or AraLAM in the presence of LPS (10 ng/ml). The specificity was determined in the presence of blocking antibodies against DC-SIGN (AZN-D2; 20 μg/ml). Supernatants were harvested after 18 hours and the IL-10 production was measured by ELISA Values are the means±standard deviations of triplicate determinations. One representative experiment out of three is shown.
b) M. bovis BCG infection of immature DC induces I-L10 production. Immature DC were infected with M. bovis BCG (MOI 4), and the experiment was performed as described in
a) ManLAM does not induce activation of immature DC. Immature DC were incubated with ManLAM, AraLAM or LPS for 18 hours, and activation was determined by measuring the expression of CD80, CD86, CD83 and HLA-DR. Dotted lines represent isotype controls, the thin lines indicate expression levels of immature DC, and the thick line represents immature DC that have been treated with either LPS (10 ng/ml), ManLAM (15 μg/ml) or AraLAM (15 μg/ml). One representative experiment out of three is shown.
b) LPS-induced activation of DC is blocked by ManLAM. Immature DC were co-cultured with LPS alone, or together with either ManLAM or AraLAM for 18 hours. Dotted lines represent isotype controls. Thick lines, and the mean fluorescence values in the histograms, represent the expression levels after treatment with LPS alone, or in combination with either ManLAM or AraLAM. Thin lines indicate the presence of antibodies against DC-SIGN throughout the incubation. One representative experiment out of three is shown.
a) M. bovis BCG induces maturation of immature DC. Immature DC were incubated with LPS or viable M. bovis BCG (MOI 4, 20 and 100) for 18 hours, and activation was determined by measuring the expression of CD80, CD86, CD83 and HLA-DR. The mean fluorescence intensity is depicted. One representative experiment out of three is shown.
b) M. bovis BCG-induced activation of DC is blocked by ManLAM. Immature DC were infected with M. bovis BCG (MOI 4). Cells were pre-incubated with 15 μg/ml of either ManLAM or AraLAM and the expression of the markers was measured after 18 hours as described in
a) Immature human DC were cultured from monocytes in the presence of GM-CSF and IL-4. The DC express next to high levels of DC-SIGN, the MR, CD83, CD86, CD80 and HLA-DR as determined by FACSscan analysis
b) DC-SIGN, expressed by immature DC, shows a similar binding to SEA as to HIV-1 gp120, a previously defined ligand of DC-SIGN. The adhesion to both SEA and HIV-1 gp120 was determined using the fluorescent bead adhesion assay. Mannan and anti-DC-SIGN MAb AZN-D1 (20 μg/ml), but not anti-MR MAb clone 19, block adhesion of SEA to the DC. One representative experiment out of three is shown, using anti-LDN antibodies to couple SEA to the fluorescent beads. Similar results are obtained using anti-LDN-DF antibodies (not shown).
a) polyacrylamide gel with SEA (15 μg) silver-stained to detect all proteins
b) Western blot of polyacrylamide gel after SDS-PAGE of SEA (2 μg), using soluble DC-SIGN-Fc to detect DC-SIGN binding glycoproteins.
a) SEA were defucosylated by mild acid treatment or α3,4-fucosidase. Binding of soluble DC-SIGN-Fc to defucosylated SEA (coated at 1 μg/ml) was measured by an anti-IgG-Fc ELISA. The degree of defucosylation, and the integrity of non-fucosylated glycans after the treatments was established using different anti-glycan MAbs, and goat-anti-mouse IgM-peroxidase for detection.
b) Competitive inhibition of the binding of soluble DC-SIGNFc to SEA and HIV gp120 by anti-glycan mAbs in ELISA. Coated antigens were preincubated with anti-glycan mAbs before adding DC-SIGNFc. Binding of DC-SIGN Fc was measured by an anti-IgGFc ELISA. Antibodies used preferably SMLDN1.1 or SMFG4.1 (anti-Lex) or SMLDN1.1 (anti-LDNF) or anti-DC-SIGN (AZN-D1, D2, AZN-D3),
c) DC-SIGN stongly binds to neoglycoproteins carrying α1,3-fucosylated oligosaccharides Lex and LDN-F but poorly to neoglycoconjugates carrying a single a-linked fucose or to LDN-DF. Neoglycoproteins were coated at a concentration of 5 μg/ml. No binding was observed to neoglycoproteins carrying Galb1,4GlcNAc or GalNAcb1,4GlcNAc (LDN) (not shown).
a) Amino acid sequence alignment of part of the CRDs of DC-SIGN (AAK20997) with that of the highly homologous C-type lectin L-SIGN (AAK20998). The position of the E324A, E347Q and V351G mutations in DC-SIGN are indicated by an arrow.
b) Binding of SEA and Lex-PAA coated beads to K562 transfectants expressing wild-type DC-SIGN and the E324A, E347Q and V351G DC-SIGN mutants, was measured using the fluorescent bead adhesion assay. To detect SEA, fluorescent beads coupled to the LDN-DF MAb were used. One representative experiment out of two is shown.
c) SEA blocks binding of DC-SIGN, expressed by human immature DC, to ICAM-3 and HIV-1-gp120. In contrast, RNAseB does not block this binding. The adhesion of both ICAM-3 and HIV-1 gp120 to DC was determined using the fluorescent bead adhesion assay. Inhibitors were present at a concentration of 20 μg/ml.
d) DC-SIGN, expressed by K562 transfectants, binds strongly to both SEA and HIV-1 gp120, whereas L-SIGN, expressed by K562 transfectants only recognizes HIV-1 gp120. The adhesion was determined using the fluorescent beads adhesion assay. To detect SEA, both anti-LDN and anti-LDN-DF coated fluorescent beads were used with similar results. One representative experiment out of two is shown, using anti-LDN-DF fluorescent beads.
e) K562 transfectants efficiently express DC-SIGN, L-SIGN and the different mutant DC-SIGNs, as determined by FACSscan analysis
A. Both mannose as well as Lewis-X epitopes are highly expressed on glycans expressed by granulocytes
B. DC-SIGN binds strongly to freshly isolated granulocytes. Freshly isolated granulocytes are incubated with DC-SIGN-Fc-coated fluorescent beads. The adhesion is determined by the fluorescent bead adhesion assay. Specificity is determined by measuring the adhesion in the presence of mannan, EGTA and antibodies against DC-SIGN (AZN-D1,D2 or D3), b2 integrins (NKI-L19) and Lex (6H3)
A. Granulocytes are surface labelled with biotine. The cell lysate is incubated over night at 4° C. with Protein A beads coated with DC-SIGN-Fc or control antibodies against ICAM-2, ICAM-3 and LFA-1. The beads are washed and the immuno-precipitated product is analysed on SDS-PAGE and visualised by autoradiograph. In particular a protein size 66 kD is immuno-precipitated. B. Granulocytes highly express the Lex containing CD66acd and CD66d antigen, do not express the DC-SIGN ligand ICAM-2 and express ICAM-3. C. Immuno-precipitation of CD66acd demonstrates strong binding activity for DC-SIGNFc. Anti-CD66acd mAbs coated on an ELISA plate were incubated with a lysate of granulocytes. After washing off non-specific proteins DC-SIGNFc was incubated to demonstrate high affinity ligands. DC-SIGNFc does not recognize ICAM-3 from granulocytes demonstrating that on granulocytes not ICAM-3 but CD66acd is the high affinity ligand for DC-SIGN. Binding of CD66acd to DC-SIGNFc is completely inhibited by removal of cations by EDTA.
293T cells are transfected with pIG-CD66a-Fc, pIG-ICAM-3-Fc and pIG (mock). Supernatants are harvested and beads are coated with the Fc chimeras (CD66a-Fc, ICAM-3-Fc and mock). Immature DC and DC-SIGN-transfected K562 cells are incubated with beads and the adhesion is determined in the presence of antibodies against DC-SIGN and P2 integrins, and mannan and EGTA.
NK cells are surface labelled with radioactive iodine. The cell lysate is incubated over night at 4° C. with Protein A beads coated with DC-SIGN-FC or control antibodies against ICAM-2, ICAM-3 and LFA-1. The beads are washed and the immuno-precipitated product is analysed on SDS-PAGE and visualised by autoradiography.
A. DC-SIGN transfectants are incubated with HSV envelope glycoprotein-coated beads and DC-SIGN binding is determined in the presence of antibodies against DC-SIGN.
B L-SIGN strongly binds HSV envelope glycoprotein gB.
L-SIGN transfectants are incubated with HCV envelope glycoprotein-coated beads and DC-SIGN binding is determined in the presence of antibodies against DC-SIGN.
B. DC-SIGN interaction with HCV E1 and E2 is concentration dependent. Mammalian cell produced E1 and E2 were titrated (0-16 or 14 mM resp.) and DC-SIGN-Fc binding was tested as described above. AZN-D1 (50 mg/ml) was used to specifically block the interaction.
C. DC-SIGN has a similar affinity for E1, E2 and gp120. Mammalian cell produced E1 and E2 (10 nM) and gp120 (2 nM) were coated and DC-SIGN-Fc binding was tested as described above. Mannan was titrated (0-1000 mg/ml) to block the interaction. Binding was expressed as percentage of the binding without block.
D. DC-SIGN binding to E1, E2 and gp120 is equally dependent on calcium. Mammalian cell produced E1 and E2 (10 nM) and gp120 (2 nM) were coated and DC-SIGN-Fc binding was tested as described above in the presence of a varying of calcium (0-5 mM). Binding was expressed as percentage of binding with 5 mM calcium.
(a-c) H. pylori α3-fucosyltransferase mutants (a), strains (b) and phase variants (c) were coated and binding of recombinant DC-SIGN-Fc was measured after incubation with peroxidase-labeled goat-anti-human Fc. H. pylori were coated and incubated with antibodies specific for Lewis antigens as indicated and their serotype was determined after incubation with peroxidase-labeled anti-mouse immunoglobulins.
(a) Lex/y positive phase variants of J223 were detected by colonyblotting with mAbs specific for the indicated Lewis antigens, after short time culturing directly from the biopsy, followed by one single passage in fluid phase and distribution over solid media. (b) C-tract sequencing was performed to determine the “on” and “off”-status of genes futA (HP0379) and futB (HP0651). The J223.3 futB mutant was generated by natural transformation with construct containing a chloramphenicol resistance marker cassette inserted in gene HP0651, and serotyped as indicated for J223. (c) Consequences of the C-tract length for functional expression of futa and futB.
(a) A tissue section of stomach was fixed and stained with anti-DC-SIGN antibodies. Original magnification×20. Arrows indicate DC-SIGN positive DC-like cells in the lamina propria. (b-d) Monocyte derived DCs (b), monocyte-derived DCs and RAW macrophages (c) or K-562 cells transfected with DC-SIGN (d) were incubated with FITC labelled H. pylori J223.3 or J223.8 and binding was analyzed using flowcytometry. In b and d, cells were preincubated with anti-DC-SIGN antibodies, mannan, EDTA, or anti-mannose receptor (MR) antibodies.
(a) DC were incubated with H. pylori J223.3 or J223.8 at an multiplicity of infection (M.O.I.) of 20 in the presence or absence of anti-DC-SIGN antibodies for 1 hr, washed and cultured for 20 hrs. Supernatant was harvested and the amount of IL-10 and IL-12p40 was analyzed by ELISA.
(b) Upon coculture of DC with H. pylori J223.3 or J223.8, cells were incubated with CD40L-transfected J558 fibroblasts in the absence or presence of IFN-γ for 24 hrs. Supernatant was harvested and the amount of IL-12p70 was analyzed by ELISA.
After preincubation with anti-DC-SIGN antibodies, DC were incubated with H. pylori J223.3 or J223.8 at an M.O.I. of 10 for 48 hrs, washed and subsequently cocultured with highly purified CD45RA+CD4+T cells. Quiescent T cells were re-stimulated with PMA and ionomycin and IL-4 and IFN-γ was analyzed on a single-cell basis by intracellular flowcytometry.
Generation of iDC and maturation conditions are described in the legend to
(A) Stable transfected HEK 293 cells with either TLR2 or TLR4 were seeded at 0.2×106 cells/well and stimulated with 107 bacteria/ml or TNFa . After 24 hours, supernatant was taken and IL-8 content was analyzed by ELISA. (B) HEK 293 cells were transiently transfected with human TLR1, TLR2, TLR3, TLR4+MD2, TLR6, TLR7 and TLR9, together with an ELAM-luciferase reporter construct and were stimulated with 107 bacteria or TNFa. Luciferase activity was determined 6 h after stimulation.
(A). Immature DC were incubated with FITC-labeled bacteria. After washing, the cells were analyzed by flow cytometry. (B) Similar protocol, as described in the part A, was used to analyze bacteria binding to DC-SIGN-transfected K562 or untransfected K562 cells. Preincubations were performed with either TSM+0.5% BSA, EDTA or anti-DC-SIGN (AZN-D1 and AZN-D2, 20 μg/ml). (C) DC-binding was performed as described in part A. Preincubations were performed with either TSM+0.5% BSA, anti-DC-SIGN (AZN-D1 and AZN-D2, 20 μg/ml), anti-MR, Mannan or methylglucoside.
Generation of iDC is described in the legend to
Polyacrylamide glycoconjugates of Lewisx and sialylated Lewisx were coated and adhesion of DC-SIGN-FC was measured with peroxidase labeled goat anti-human Fc in DC-SIGN-Fc ELISA. B. Lewisx expression was determined on PMN labeled for CD16 to discriminate between CD16+ neutrophils and CD16− eosinophils. C. The adhesion of beads coated with ICAM-3-Fc or DC-SIGN-Fc to PMN was assessed using flow cytometry. Specificity of adhesion was obtained using blocking anti-DC-SIGN antibodies (AZN-D1, 20 mg/ml), mannan (50 mg/ml), or EGTA (10 mM). D. Titration of DC-SIGN-Fc to PMN reveals high affinity binding of DC-SIGN to PMN. E. Binding of optimal concentration of DC-SIGN-Fc (10 mg/ml) to PMN labeled for the neutrophil marker CD16. To assess specificity blocking anti-DC-SIGN antibodies (AZN-D1, 50 mg/ml) were used.
A Capture of CD11b and CD66acd from PMN lysate using specific antibodies revealed the presence of Lewis X antigens on these cell membrane molecules
B Capture of CD11b and CD66acd from PMN lysate and coupling to fluorescents beads revealed that in particular PMN-derived CD11b and CD66acd interacted strongly with DC-SIGN expressing transfectants indicating that DC-SIGN binds PMN derived CD11b and CD66acd.
C Captured CD11b/CD18 and CD66 out of PMN lysate demonstrates high affinity binding with DC-SIGNFc as demonstrated in DC-SIGN Fc ELISA.
D. Immunoblotting studies demonstrate that DC-SIGNFc specifically interacts with CD66acd and CD11b from PMN when immunoprecipitated (IP) using anti-CD66acd (lane 9) and CD11b mAbs (lane 10) out of a PMN lysate. Cell-surface proteins are biotinylated and PMNs are lysed. Lane 1. negative control using ICAM-3 Fc to capture by IP proteins. Detection is peroxidase-labeled streptavidin. Lane 2. DC-SIGN-Fc is used to capture ligands biotinylated ligands from lysate; two protein bands are visible, the upper band consists of CD11b and CD66a, whereas the lower consists of CD18. Lane 3. Negative control; no bands are immune precipitated with anti-DC-SIGN antibodies. Lane 4, IP with antibodies against CD66acd; a faint band is visible containing CD66a. Lane 5, IP of CD11b; two bands are visible; the upper band is CD11c and the lower band is CD18m, which is co-immuneprecipitated with CD11c. Lane 6. IP with DC-SIGN ligands and detection with anti-CD66acd; CD66a is captured by DC-SIGN Fc. Lane 7. IP with DC-SIGN ligands and detection with anti-CD11b; CD11b is captured by DC-SIGN Fc. Lane 8. Negative control, IP with anti-DC-SIGN antibodies and detection with DC-SIGN-Fc. Lane 9. IP with anti-CD66acd and detection with DC-SIGN Fc; CD66a is bound by DC-SIGN on blot. Lane 10. IP with anti-CD11a and detection with DC-SIGN Fc; CD11a is bound by DC-SIGN on blot.
A. DC-SIGNFc recognizes tumor antigens on several breast carcinomas and adenocarcinomas. Binding can be blocked by anti-DC-SIGN mAbs
B. the tumor cell line SW948 interacts with DC-SIGN expressing cells and with DCs and the cell adhesion can be blocked by anti-DC-SIGN antibodies (AZN-D1), indicating a function for DC-SIGN to interact with a cellular structure on tumor cells
C. DC-SIGN-FC binds immuneprecipitated CD66e (CEA). Immunoprecipitation with anti-CD66e (anti-CEA) of cell-surface biotinylated SW948 tumor cell-lysate shows that DC-SIGN-FC binds the tumor antigen CEA (lane 5).
A. CD66e from the tumor cell line SW948 expresses Lewis X and Lewis Y carbohydrate structures s determined using specific antibodies.
B. Captured CD66e from SW948 lysate is bound by DC-SIGN-Fc
C. DC-SIGN-FC captures CD66e from a SW948 lysate as detected by an anti-CD66e specific antibody.
D. Immunoprecipitation (IP) studies revealed that binding of DC-SIGN-Fc to CD66e is abrogated when CD66e is treated with a-1,3,4 Fucosidase that removes the fucose residue present in Lewis X,Y (Lane 3 and 4).
Methods.
Antibodies and proteins. The following monoclonal antibodies (Mab) were used: anti-CD107a (Lamp-1; MabH4A3, BD Pharmingen)., anti-MR (Clone 19, BD Pharmingen), CD11b (bear-1)25, CD11c (SHCL3)26, anti-DC-SIGN (AZN-D1, AZN-D214, CSRD10, blocking CD11a (NKI-L15), anti-ICAM-1 (Rek-1), anti-ICAM-2(12A2), and anti-ICAM-3 (AZN-IC3/1, icr-2), activating CD18 (KIM185) and the PE/FITC-conjugated antibodies CD25, CD69, CD80, CD86, HLA-DR (BD Pharmingen), and CD83 and CD56 (Beckman Coulter). Biotin-conjugated CD3 and CD16 (BD Pharmingen). CLB-T4 (αCD4) was a kind gift of René van Lier. MEM-157 (αCD16) and MEM-97 (αCD20) were kindly provided by Vaclav Horejsi. ICAM-1 mAb CBR-IC-1/3, ICAM-2 mAb CBR-IC-2/1, and ICAM-3 mAb CBR-IC-3/2 were obtained through the Human Leukocyte Differentiation Antigen Workshop. Several anti-glycan monoclonal antibodies (MAbs) were used: the anti-LDN-DF MAb 114-5B1-A16, the anti-LDN MAb SMLDN1.117, the anti-LDN-F MAb SMLDNF110 and the anti-Lex MAb CB1027, the anti-Lex mAbs SMLDN1.1 and SMFG4.1 and 6H3 (anti-Lex). The mAb (CLB-gran/10) was used to stain and immunoprecipitate CD16.
The neoglycoprotein HSA-Lex, containing approx. 20-25 oligosaccharide chains per HSA molecule, was from Isosep A B, Tullinge, Sweden. The neoglycoprotein BSA-LDN-DF (approx. 12 oligosaccharide chains per molecule BSA), and BSA-LDN-F (approx. 3-4 oligosaccharides per molecule BSA), were synthesized enzymatically as described by van Remoortere et al16, and Nyame et al17, respectively. Lex-PAA-biotin, containing Lex multivalently coupled to biotinylated polyacrylamide was from Syntesome, Munich, Germany.
Mannan purified from Saccheromyces cerevisiae (50 μg/ml) and recombinant gp120 (0.50 μg/ml) were obtained from Sigma and the Aidsresource Foundation, respectively. Purified mannose capped lipoarabinomannan (manLAM) from Mycobacterium tuberculosis, and non-capped LAM (araLAM) from M. smegmatis were kindly provided by Dr. J. Belisle, Colorado State University through NIH, NIAID contract NO1 AI-75320. Soluble egg antige (SEA) was kindly provided by Dr. A. K. Nyame, Oklahoma University HSC, USA). Purified lipophosphoglycan from Leishmania mexicana was kindly donated by Dr. M Wiese, Bernard Nocht Inst. Tropical Medicine Hamburg, Germany. Purified lipopolysaccharide of Helicobacter pylori was obtained from M. Monteiro, NRC, Ottawa, Canada. A sonicate of bacterial cells of a clinical isolate of M. tuberculosis was donated by A. Kolk, KIT, Royal Trop. Inst, Amsterdam. Clinical isolates of Helicobacter pylori, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus were obtained from VUMC Hospital, Amsterdam. Synthetic glycoconjugates were obtained from Syntesome, Munich, Germany, and comprise mono- and oligosaccharides multivalently linked to a biotinylated polyacrylamide carrier, MW 40.000.
Soluble DC-SIGN-Fc adhesion assay. DC-SIGN-Fc consists of the extracellular portion of DC-SIGN (amino acid residues 64-404) fused at the C-terminus to a human IgG1-Fc fragment. DC-SIGN-Fc was produced in Chinese Hamster Ovary K1 cells by co-transfection of DC-SIGN-Sig-pIgG1 Fc (20 μg) and pEE14 (5 μg) vector. DC-SIGN-Fc concentrations in the supernatant were determined by an anti-IgG1 Fc ELISA. The DC-SIGN-Fc binding assay was performed as follows. Glycoconjugates and sonicated mycobacteria were coated onto ELISA plates at 5 μg/well; intact bacterial cells were coated at 5×107/ml; coating took place for 18 hours at room temperature, followed by blocking with 1% BSA for 30 min, at 37° C. in TSM (20 mM Tris-HCl pH 7.4 containing 150 mM NaCl, 2 mM CaCl2 and 2 mM MgCl2). Soluble DC-SIGN-Fc (approx. 2 μg/ml in TSM buffer) was added and the adhesion was performed for 120 min. at RT. Unbound DC-SIGN-Fc was washed away and binding was determined by an anti-IgG1 Fc ELISA using a peroxidase conjugate of goat anti-human Fc. Specificity was determined in the presence of either 20 μg/ml blocking antibodies, 50 μg/ml mannan or 5 mM EGTA.
Cells. Immature DC were cultured from monocytes in the presence of IL-4 and GM-CSF (500 and 800 U/ml, respectively; Schering-Plough, Brussels, Belgium28. At day 7 the phenotype of the cultured DC was confirmed by flow cytometric analysis. The DC expressed high levels of MHC class I and II, αMβ2 (CD11b), αXβ2 (CD11c) and ICAM-1, moderate levels of LFA-1 and CD80, and low levels of CD14. K562 transfectants expressing wild-type DC-SIGN were generated by transfection of K562 cells with 10 μg pRc/CMV-D C-SIGN plasmid by electroporation as previously described.
Stable transfectants of K562 expressing ICAM-3 were obtained by electroporation of pCDM8-ICAM-3 and pGK-HYG. K562 cells were cultured on RPMI 10% FCS, whereas K562-ICAM-3 cells were cultured on RPMI 10% FCS: Iscove's 5% FCS 3:1 containing 0.5 mg/ml hygromycine to maintain ICAM-3 expression.
Naive NK cells were isolated from buffy coats of healthy donors. Briefly, the PBMC fraction obtained through Ficoll centrifugation was sequentially depleted for CD14+ cells, and CD3+, CD4+, and CD20+ cells using MACS sorting. CD14+ cells were depleted by CD14 microbeads (Miltenyi Biotec) on an LS column (Miltenyi Biotec), and CD3+, CD4+, and CD20+ cells were labeled by the mAbs T3B (αCD3), CLB-T4 (αCD4), MEM97 (αCD20), and thereafter depleted by goat-α-mouse microbeads (Miltenyi Biotec) on an LD column (Miltenyi Biotec). Thus obtained NK cells were routinely tested for the NK cell markers CD16 (75% to 90% expression), CD56 (80 to 95% expression), the non-lineage markers CD3, CD4, CD14 and CD20 (all less than 1% expression), and the early activation marker CD69 (15% expression on non-activated NK cells and 75% expression on 1-day IL-2-activated NK cells). CD56dim and CD56bright NK cell populations were isolated from MACS obtained NK cells by FACS sorting on low and high CD56 expression, respectively. CD56dim and CD56bright NK cell populations were over 95% pure as assessed by flow cytometry staining for CD16 and CD56.
DC activation. Immature DC (2×106 cells/ml) were cultured for 24 hours in the presence of IL-4 (500 U/ml, Schering-Plough, Brussels, Belgium), GM-CSF (800 U/m; Schering-Plough, Brussels, Belgium) and either LPS (10 ng/ml) or LAM glycolipids (15 μg/ml). The effect of LAM on LPS-induced activation was determined by pre-incubating immature DC (300.000 cells) with AZN-D2 (40 μg/ml) for 30 minutes, and subsequently with LPS in the presence of LAM (15 μg/ml) for 18 hours. LAM glycolipids were obtained from J. Belisle (Colorado State University and the NIH (contract NO1 AI-75320)) and contained <5 ng/mg endotoxin. Activation was determined by cell-surface expression of MHC class II (HLA-DR) and the co-stimulatory molecules CD80, CD83 and CD86 using PE-conjugated antibodies.
Fluorescent bead adhesion assay. Carboxylate-modified TransFluorSpheres (488/645 nm, 1.0 μm; Molecular Probes, Eugene, Oreg.) were coated with HIV-1 gp120 and ICAM-3 as described14. Streptavidin was covalently coupled to the beads as described and streptavidin-coated beads were incubated with biotinylated PAA-linked glycoconjugates (50 pMol; Syntesome, Munich, Germany). The fluorescent bead adhesion assay was performed as described14. Ligand-coated fluorescent beads (20 beads/cell) were added to the cells for 45 minutes at 37° C., washed and analyzed by flow cytometry (FACScan, Becton Dickinson, Oxnard, Calif.), by measuring the percentage of cells that had bound fluorescent beads. LAM-coated beads were generated by coating incubating streptavidin-coated beads were incubated with biotinylated F(ab′)2 fragment goat anti-mouse IgG (6 μg/ml; Jackson Immunoresearch) followed by an overnight incubation with mouse-anti-LAM antibody (F30.5) at 4° C. The beads were washed and incubated with 250 ng/ml purified glycolipid LAM (obtained from J. Belisle, Colorado State University and the NIH (contract NO1 AI-75320)) overnight at 4° C. SEA-coated beads were generated by incubating the streptavidin-coated beads with biotinylated F(ab′)2 fragment of goat anti-mouse IgG (6 μg/ml; Jackson Immunoresearch), followed by an overnight incubation at 4° C. with anti-LDN MAb, or anti-LDN-DF MAb. The beads were washed and incubated with 1 μg/ml SEA overnight at 4° C. Essentially, 50×103 cells were pre-incubated in adhesion buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM CaCl2, 2 mM MgCl2, 0.5% BSA) with or without blocking MAbs (20 μg/ml) or mannan (50 μg/ml) for 10 minutes at room temperature. Ligand-coated fluorescent beads (20 beads/cell) were added to the cells and the suspension was incubated for 45 minutes at 37° C. Cells were washed and adhesion was determined using flow cytometry (FACScan, Becton Dickinson, Oxnard, Calif.), by measuring the percentage of cells that had bound fluorescent beads. HIV-1 gp120 fluorescent beads were prepared as described previously19.
DC-SIGN-Fc adhesion. Cells were incubated with DC-SIGN-Fc for 30 minutes at 37° C. under saturating conditions (concentration: 10 μg/ml), and subsequently with FITC-conjugated goat-α-human secondary antibodies to monitor adhesion of DC-SIGN-Fc. Before incubation with cells, DC-SIGN-Fc was preincubated for 10 min at RT either with medium, aDC-SIGN (AZN-D1, 50 μg/ml), mannan (50 μg/ml), or EGTA (10 mM) to determine specificity of DC-SIGN-Fc adhesion. DC-SIGN-Fc adhesion was determined by flow cytometry (FACS Calibur, Beckman Coulter).
Mycobacteria. Both the M. bovis BCG (Pasteur) and the M. tuberculosis H37Ra strains were gifts from A. Kolk (Royal Tropical Institute, Amsterdam). M. bovis BCG was cultured in vitro using Middelbrook 7H9 broth supplemented with 0.05% Tween 80 and albumin-dextrose-catalase. The glycolipids ManLAM and AraLAM were obtained from J. Belisle, Colorado State University and the NIH (contract NO1 AI-75320). DC were infected with mycobacteria by co-culturing them at an appropriate multiplicity of infection (MOI) as indicated in the figure legends.
Fluorescent mycobacterial binding assay. Capture and internalization of mycobacteria by cells was evaluated using fluorescein isothiocyanate (FITC)-conjugated M. bovis BCG. Bacteria (109/ml) were labeled by incubation of 0.5 mg FITC per ml in phosphate buffered saline (pH 7.4) at room temperature for 1 hour. The FITC-pulsed bacteria were washed three times to remove unbound FITC. Capture was determined by measuring the percentage of cells that bound FITC-conjugated bacteria using flow cytometry (FACScalibur, Becton Dickinson Immunocytometry, San Jose, Calif.). Phagocytosis was determined using a fluorescence-quenching technique as reported previously29. In brief, quenching of non-internalized membrane-bound FITC-conjugated M. bovis BCG was achieved by treating the cells with 0.05% trypan blue for 5 minutes. H. pylori binding was assessed by labeling the bacteria with FITC and binding to DC was investigated similarly to the bead assay.
Cytokine production. For the detection of cytokines, culture supernatants were harvested at day 1 and frozen at −80° C. until analysis. The supernatants were analyzed for the presence of IL-10 and IL-12p40 by ELISA (Biosource International, Calif.).
Defucosylation of SEA. SEA were defucosylated by incubation of the antigens at 100° C. for 1 hour in 0.1M TFA. After neutralization, the defucosylated antigen was coated to ELISA plates. Enzymatic defucosylation was performed by incubating the SEA in 50 mM sodiumphosphate pH=5.0 with a1,3/4-fucosidase (0.4 mU/μg SEA) (Calbiochem) overnight at 37° C. The degree of defucosylation of the antigens was assessed by their ability to bind MAbs specifically recognizing the fucosylated glycan epitopes LDN-DF and Lex, whereas the integrety of other, non-fucosylated, glycan epitopes was assessed by measuring the reactivity with anti-LDN MAb.
SDS-PAGE, Western blotting and silver staining. SEA were separated by SDS-PAGE under reducing conditions on a 12.5% polyacrylamide gel, using the Mini-Protean II system (BioRad), and proteins visualized by silver-staining. For Western blotting proteins were transferred onto a nitrocellulose membrane (Schleicher and Schuell). The membrane was blocked in a solution of 5% BSA in TSM for 2 h followed by incubation in 2 μg/ml DC-SIGN-Fc in TSM buffer containing 1% BSA for 1 h. After washing, the membrane was subsequently incubated for 1 h in peroxidase conjugated goat anti-human IgG1 and reactive bands were visualized by detection with CN/DAB substrate (Pierce).
Cytotoxicity. The standard 4-hour 51Cr release assay was used to assess NK cell-mediated cytotoxicity. Briefly, 1*106 target cells were labeled by 100 μCi 51Cr for 1 hour at 37° C., extensively washed to remove free 51Cr, resuspended at 2500 cells/well (iDC and mDC) or 1000 cells/well (K562 and K562-DC-SIGN), and incubated with NK cells for 4 hours at 37° C. at the indicated ratios and under the indicated conditions. After 4 hours scintillation liquid (PerkinElmer) was added to supernatants, and 51Cr release was determined on a micro-b counter (PerkinElmer).
NK cell-mediated DC maturation. Resting and activated NK cells were obtained by overnight incubation on medium and IL-2 (1000 U/ml), respectively. Thereafter, resting and activated NK cells were incubated overnight with immature DC in a 96 wells U-bottom plate (Costar) on RPMI 10% FCS. NK cells were preincubated for 10 min. at RT with medium or blocking αLFA-1 mAbs (NKI-L15, 50 μg/ml), whereas DC were preincubated with medium or blocking αDC-SIGN mAbs (AZN-D2, 50 μg/ml). As a positive control DC were incubated overnight in the presence of LPS (2 μg/ml). Maturation of DC was assessed by flow cytometry (FACS Scan, Beckman Coulter) for the maturation markers CD80, CD83, CD86, and HLA-DR by PE-conjugated or FITC-conjugated mAbs.
DC-induced NK cell activation. Resting NK cells were incubated for 2 days with immature or mature DC (obtained by LPS maturation) in a 96 wells U-bottom plate (Costar) on RPMI 10% FCS. LFA-1-dependent DC-induced NK cell activation was determined by a 10 min. preincubation of NK cells with αLFA-1 mAbs (NKI-L15, 50 μg/ml), whereas dependency on DC-SIGN was assessed by a 10 min. preincubation of DC with anti-DC-SIGN mAbs (AZN-D1 and AZN-D2, 50 μg/ml). As a positive control NK cells were incubated for 2 days with IL-2 (1000 U/ml). NK cell activation was assessed by flow cytometry (FACS Calibur, Beckman Coulter) for the early activation marker CD69 by FITC-conjugated mAbs.
Immunoprecipitation. NK cells were surface iodinated with 1 mCi 125I, or biotinylated and subsequently lysed in lysisbuffer (1% Triton-X-100, 10 mM TEA, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM PMSF, 20 μg/ml trypsin inhibitor, 20 μg/ml leupeptin, and 20 μg/ml aprotinin). DC-SIGN ligands, ICAM-2, ICAM-3-, and LFA-1 were immunoprecipitated from prot A-precleared NK, cell lysate by prot A beads covalently linked to DC-SIGN-Fc, αICAM-2 (12A2), αICAM-3 (AZN-IC-3/1), and αLFA-1 (NKI-L15). Immunoprecipitates were reduced in sample buffer (containing 4% SDS and 5% b-mercapto-ethanol), heated for 5 min at 95° C., and run on a 5-15% gradient polyacrylamide gel (SDS-PAGE).
Alternatively, specific anti-bodies against CD66acd or ICAM-3 or LFA-1 were coated in an ELISA plate, subsequently lysates were added and incubated for 1 hr at 4 C to retract the specific proteins. DC-SIGNFc was incubated for binding activity to the specific proteins and stained with Goat anti human Fc PO and developed.
Experimental Setup.
Dendritic cells (DC) are instrumental in handling pathogens for processing and presentation to T cells, thus eliciting an appropriate immune response. C-type lectins expressed by DC function as pathogen-recognition receptors; yet their specificity for carbohydrate structures on pathogens is not fully understood. Here we analyzed the carbohydrate specificity of DC-SIGN/CD209, the recently documented HIV-1 receptor on DC. Our studies show that DC-SIGN binds with high affinity to both synthetic mannose- and fucose-containing glycoconjugates. These carbohydrate structures are abundantly expressed by pathogens as demonstrated by the affinity of DC-SIGN for natural surface glycans of the human pathogens Mycobacterium tuberculosis, Helicobacter pylori, Leishmania mexicana and Schistosoma mansoni. Strikingly, these pathogens target DC-SIGN to infect DC but also to modulate the immune responses mediated by DC.
This analysis expands our knowledge on the carbohydrate and pathogen-specificity of DC-SIGN, and identifies this lectin to be central in pathogen-DC interactions.
For in vitro binding studies we generated a chimeric protein of DC-SIGN with a human IgG1 Fc tag, which we used to screen in an ELISA format for reactivity with a panel of synthetic glycoconjugates containing mannose or fucose residues and their derivatives in multimeric form.
As reported earlier, DC-SIGN-FC binds to purified yeast-derived mannan and the high mannose containing HIV-1 gp120, but also to less complex mannose-containing glycoconjugates i.e. mannose and α1→3, α1→6 mannotriose (Table I,
To determine whether DC-SIGN-FC exhibits a similar carbohydrate recognition profile as cell-surface expressed DC-SIGN, both DC-SIGN transfectants and monocyte-derived DC were studied for carbohydrate binding activity using a fluorescent beads adhesion assay with different glycoconjugates (α1→3, α1→6mannotriose, Lex and sulfo-Lea) (
We subsequently investigated the binding of DC-SIGN to human pathogens that express mannose- or fucose-containing glycans. The gram-negative Helicobacter pylori bacterium, which induces peptic ulcers and gastric carcinoma20, and the worm parasite (the causal agent of schistosomiasis) both express Lex 21. In H. pylori, Lex is present on surface-located lipopolysaccharide, while in S. mansoni Lex is expressed by all stages of the parasite, including soluble egg antigen (SEA)21. Binding of DC-SIGN-Fc to Lex-positive H. pylori lysate and to extract of S. mansoni was strong and was completely inhibited by anti-DC-SIGN antibodies (
Whole mycobacteria such as the M. tuberculosis H37Ra strain and M. bovis bacillus Calmette-Guérin (1BCG) were coated and the interaction of DC-SIGN with these pathogens was analyzed using the DC-SIGN-Fc binding assay. M. bovis BCG is a tuberculosis strain that is almost nonpathogenic yet retains the immunological properties of tuberculosis. DC-SIGN-Fc interacted specifically with both M. tuberculosis H37Ra and M. bovis BCG, since the interaction was inhibited with blocking DC-SIGN-specific antibodies (
Moreover, an irrelevant Fc chimera, ICAM-3-Fc, did not interact with the mycobacteria (
We next investigated the binding of DC-SIGN to purified mycobacterial lipoarabinomannan (LAW, since DC-SIGN has a high affinity for mannose-containing carbohydrates and LAM is the major mannose-containing component of the mycobacterial cell-wall31. LAM comprises a mannose-rich polysaccharide-core, containing highly branched arabinofuranosyl side chains, and a GPI anchor (
We used K562 transfectants stably expressing DC-SIGN to investigate the binding of cell-surface-expressed DC-SIGN to both M. bovis BCG and the mycobacterial component ManLAM. These cells do not express the previously reported mycobacterial receptors Mannose Receptor (MR), CD11b and CD11c (
The C-type lectin domain of DC-SIGN contains two calcium ions, and the amino acid residues that are in close contact with Ca2+ at site 2 (Glu347, Asn349, Glu354 and Asn365) form the core of the ligand binding site23. Changing in DC-SIGN either Glu347 into Gln (E347Q), or Asn349 and Asn365 into Asp, resulted in complete loss of binding to whole mycobacteria and ManLAM (
Recently, we demonstrated that the binding site of DC-SIGN for its cellular ligand ICAM-3 is distinct from that of HIV-1 gp120, since a specific mutation in DC-SIGN (V351G) abrogated ICAM-3, but not HIV-1 gp120 binding (
Immature DC express, besides high levels of DC-SIGN, high levels of the receptors MR, CD11b and CD11c (
The major contribution of DC-SIGN to the interaction of immature DC to ManLAM prompted us to investigate whether DC-SIGN could be important in the interaction of immature DC to whole mycobacteria. Immature DC strongly interacted with M. bovis BCG (
Next we investigated whether DC-SIGN mediates internalization of M. bovis BCG using trypan blue to quench surface FITC-conjugated mycobacteria. Mock transfected K562 cells did not phagocytose M. bovis BCG, whereas 50% of the K562 transfectant, expressing DC-SIGN, had internalized M. bovis BCG within 45 minutes (
Immature DC are highly phagocytosing cells and indeed within 45 minutes more than 90% of the dendritic cells that bound M. bovis BCG, had internalized the mycobacteria (
Recently, we have demonstrated that DC-SIGN can function as an antigen receptor that internalizes antigens and targets them to lysosomal compartments for presentation on MHC Class II10. Therefore, the fate of the captured M. bovis BCG by immature DC was followed by immuno-fluorescence analyses. Immature DC were incubated with FITC-conjugated M. bovis BCG for 2 hours and both DC-SIGN and Lamp-1 were stained (
Recently, we demonstrated that binding of soluble ligands or antibodies to DC-SIGN triggers internalization of the DC-SIGN-ligand complex to the late endosomes/lysosomes, and results in down-regulation of DC-SIGN from the surface10. Therefore, we investigated whether DC-SIGN is internalized after ManLAM binding by measuring the cell-surface expression of DC-SIGN after ManLAM binding using a specific antibody against DC-SIGN. Indeed, binding of ManLAM, but not AraLAM, to DC-SIGN results in down-regulation of DC-SIGN (
ManLAM is present not only a mycobacterial cell-wall component but is also secreted from phagosomes following macrophage ingestion of M. tuberculosis31. Potentially, mycobacteria within infected macrophages can influence bystander immune cells and modulate the immune response through secretion of ManLAM. The cytokine IL-10 is a potent immunosuppressive factor induced in macrophages by some intracellular bacteria to dampen down host immune responses and promote their survival32. We investigated the influence of ManLAM binding to DC-SIGN in IL-10 production by DC. ManLAM alone did not induce IL-10 production by immature DC (
Immature DC are highly efficient in antigen capture and processing, whereas mature DC are specialized in the naive T cell activation necessary for cellular immune responses. Immature DC mature in response to specific ‘danger’ signals such as bacterial components (LPS) or inflammatory cytokines (TNFa, PGE2). We investigated the effect of ManLAM on the maturation of DC. Neither ManLAM nor AraLAM induced DC maturation, since both ManLAM and AraLAM, in contrast to LPS that triggers TLR4, did not up-regulate expression of the activation markers CD80, CD83, CD86 or HLA-DR (
Acute mycobacterial infections represent sites of inflammation that attract and induce DC maturation through the presence of maturation components. Therefore, we investigated the effect of ManLAM and AraLAM in combination with the stimulatory bacterial LPS. Toll-like receptor-4 (TLR4) interaction with LPS generates intracellular signaling, most notably via the transcription factor NFkB, that results in DC activation/maturation. Indeed, DC efficiently mature in the presence of LPS alone (
Both M. tuberculosis and M. bovis BCG are able to induce DC maturation through their cell-wall components via TLR2- and TLR4-mediated signaling33-36. Indeed, M. bovis BCG infection of immature DC results in DC maturation, as demonstrated by the increased expression of MHC class II and the co-stimulatory molecules CD80, CD83 and CD86 after M. bovis BCG infection (
Because DC are central in directing Th1-Th2 responses, we searched for a cell-surface receptor expressed on DC that interacts with S. mansoni SEA. To detect binding of SEA to human immature DC, a fluorescent bead adhesion assay was developed. Fluorescent beads were pre-coated with monoclonal antibodies (MAbs) to SEA glycan antigens and then used to capture SEA. The conjugated beads were allowed to interact with DC. SEA is a mixture of glycoproteins, containing many immunogenic glycan antigens (3, 36). Major glycan antigens present in SEA, and the recognition of these antigens by anti-glycan MAbs, are depicted in
To further analyze the binding properties of DC-SIGN to S. mansoni SEA, we investigated binding of soluble chimeric DC-SIGN-Fc to SEA. In an ELISA based assay DC-SIGN-Fc showed efficient binding to wells coated with SEA (
Many glycoproteins of SEA are heavily fucosylated (3, 36). Because it has been reported that DC-SIGN may exhibit binding to both mannose and fucose, and Western blotting of SEA suggested that Lex and/or LDN-DF glycans are present on SEA proteins with similar apparent MW as the SEA proteins binding to DC-SIGN, we explored whether defucosylation of SEA affects its recognition by DC-SIGN-Fc. Treatment of SEA with the Xanthomonas □1,3/1,4fucosidase resulted in 50% decreased reactivity of anti-Lex MAb to SEA, whereas no loss in reactivity was observed, as expected, with anti-LDN-DF Mab. The LDN-DF epitope contains terminal α2-linked fucose. The results thus indicate that the that the glycosidase treatment specifically removed part of the a3-fucose moieties present on SEA. The loss in a¾-fucose residues in SEA upon treatment with the Xanthomonas α1,3/α1,4-fucosidase also resulted in a 25% loss of binding of DC-SIGN-Fc (results not shown). These data suggest that α1,3/α1,4-linked fucose residues may be important for binding of soluble DC-SIGN-FC to SEA.
Next we investigated whether one or more of the fucosylated SEA antigens Lex, LDNF or LDN-DF (see
The C-type lectin domain of DC-SIGN binds two Ca2+ ions and those amino acid residues in close contact with Ca2+ at site 2 (Glu347, Asn349, Glu354 and Asn865) or with Ca2+ at site 1 (Asp320, Glu324, Asn350 and Asp355) are essential for ligand binding37. Because the results above indicated that the α3-fucosylated trisaccharide Lex on SEA may function as a ligand for DC-SIGN, we investigated the binding properties of these antigens to K562 cell transfectants expressing mutated forms of DC-SIGN. Mutation of either E324 to Ala, or E347 to Gln in DC-SIGN (
It has recently been demonstrated that a specific mutation in the CRD of DC-SIGN (V351G) allows binding of HIV-1 gp120, but abrogates binding to ICAM-3, a T-cell ligand that has been shown previously to interact with DC-SIGN. Our results indicate that the DC-SIGN V351G mutant does not bind to either SEA or Lex, whereas binding to HIV-1 was still observed, indicating that Val351 is essential for binding to both SEA and Lex, but not to HIV-1 (
L-SIGN, an adhesion receptor that resembles DC-SIGN by recognizing ICAM-2, ICAM-3 and HIV-gp120, contains a CRD that is nearly identical to that of DC-SIGN38, and both receptors recognize high-mannose-type N-glycans. Our results above demonstrated a novel binding activity of DC-SIGN to fucosylated glycans and showed that Val35l in DC-SIGN is essential for this binding activity. At the corresponding position in the CRD of L-SIGN a Ser is present instead of Val (
DC-SIGN is known to interact with ICAM-2 and ICAM-3, however the glycan ligands on these molecules have not yet been identified. The blood group antigen Lex (CD15) is expressed by gastric mucosal epithelial cells, and by polymorphonuclear leukocytes (granulocytes) (
Moreover, we predict that based on the carbohydrate recognition pattern of DC-SIGN (
Immuno-precipitation studies revealed that DC-SIGN-Fe does not bind the cellular DC-SIGN counter-structures ICAM-2 or ICAM-3 from granulocytes but binds to a novel glycoprotein with a weight of 66 kD protein that is only present on granulocytes (
To determine whether DC-SIGN preferentially interacts with naive T cells expressing high levels of ICAM-3, as was previously postulated, we started to investigate DC-SIGN binding to total PBL population of cells. Strikingly, DC-SIGN-Fc interacted with a CD3− cell population that were CD56+ and belonged to NK cell subset (
To investigate whether the interaction of DC-SIGN with NK cells is relevant in NK mediated immune response we invested both the lysis of DC by NK cells and the maturation of DC by NK cells; two processes that have been recently identified to play an important role in innate immunity.
DC-SIGN did not inhibit DC maturation by NK cells (
Previous results demonstrated that DC-SIGN specifically interacts with HIV-139 and recently it was published that DC-SIGN binds to Ebola virus40. Therefore, we investigated whether DC-SIGN and its homologue L-SIGN can interact with other viruses, that contain glycosylated envelope proteins. Strikingly, DC-SIGN-Fc binds strongly to Herpes simplex virus (HSV)-1 and -2, and this interaction is specically inhibited by antibodies against DC-SIGN (
Materials and methods particular to this example and relevant for the experiments described in figure legends 33 to 66.
Bacterial Strains.
H. pylori clinical isolates of different geographic origins (The Netherlands, Canada, Poland, Italy, and the People's Republic of China) have been described before5, as well as H. pylori strain NCTC 11637 and phase variants 1c, K4.1, K5.1, 3a, and 1b9. H. pylori strain 4187E and generation of futA (gene HP0379) and futB (gene HP0651) knockout mutants have been described10. Clinical isolate H. pylori J223 was a obtained from Dr. H. P. Wirth6. Lex/y positive and negative variants of J223 were isolated as previously described9. Briefly, bacteria from the biopsy were inoculated on Dent plates (Columbia agar, 5% horse blood+Dent supplement), followed by one single passage in fluid phase (Brucella broth+3% fetal calf serum) and distribution over solid media, after which detection of LPS phase variants took place by colony blotting9. Genotyping by Amplified Fragment Length Polymorphism (AFLP) analysis was performed as described in reference39 with the following modifications: : In a final volume of 20 mL, 20 ng of purified chromosomal DNA was digested with 1 U of EcoRI (Pharmacia, Uppsala, Sweden) and 1 U of MseI (New England Biolabs, Beverly, Mass.). The final volume then was increased to 30 mL by addition of 50 pmol each of the EcoRI adaptor and MseI adaptor, 1.2 U of T4 DNA ligase (Pharmacia), 1 mM ATP, and ligase buffer. The adaptors were allowed to ligate to the restriction fragments for 16 h at 167 C, after which the sample was diluted with distilled water in a final volume of 500 mL. Texas Red fluorescent-labeled EcoO primer (50-AGACTGCGTACCAATTC-30; Isogen Bioscience, Maarssen, The Netherlands) and unlabeled MseO primer (50-GACGATGAGTCCTGAG-30) were used for DNA amplification in a GeneAmp PCR system 9700 thermal cycler (Perkin-Elmer) with 35 cycles: denaturation (30 s at 94° C.), annealing (30 s at 65-56° C.), and DNA molecule extension (60 s at 72° C.). In the first 12 cycles, the annealing temperature was lowered 0.7° C. per cycle. After completion of the cycle program, each sample was analyzed according to the manufacturer's instructions in a Vistra 725 automated DNA sequencer (Amersham Life Science). Fluorescent AFLP images were stored as TIFF files with the Vistra 2 TIFF software (Amersham).
Monoclonal Antibodies and Glycoconjugates.
H. pylori strains and phase variants were serotyped as previously described5 with the following monoclonal antibodies (mAbs): Hp151, specific for Ley, 6H3, specific for monomeric Lex and 4D2, specific for H type 1 (all three a kind gift from R. Negrini, General Hospital, Brescia, Italy). MAb 54.1F6A, specific for polymeric Lewis x (a kind gift from G. van Dam, Leiden, The Netherlands), NAM61-1A2, specific for i antigen (a kind gift from D. Blanchard, Regional Blood Transfusion Service, Nantes, France)10. MAbs AZN-D1 and AZN-D221 were used to block DC-SIGN and MAb Clone 17 (a kind gift from S. Gordon, University of Oxford, UK) was used to block the mannose receptor.
Synthetic glycoconjugates (Syntesome, Munich, Germany) comprised mono- and oligosaccharides that were multivalently linked to a polyacrylamide carrier. Ceramide-linked dimeric-, trimeric and tetrameric Lex antigens were a kind gift from R. R. Schmidt, University of Konstanz, Germany, and were synthesized as described40.
Cells
Immature DC were generated by culturing monocytes in RPMI-1640/10% FCS in the presence of IL-4 (500 U/ml, Schering-Plough, Kenilworth, N.J.) and GM-CSF (800 U/ml, Schering-Plough) for 5-8 days41. K-562 cells, K-562-DC-SIGN cells and RAW 264.7 macrophages were cultured as described41.
Bacterial Binding
H. pylori cells were labeled with FITC. Cells (50,000) in TSM with 0.5% BSA were preincubated with 20 ug/ml mAb, 200 ug/ml mannan or 10 mM EDTA for 10 min at RT. FITC-labeled bacteria (10 bacteria/cell) were added and incubated for 45 min at 37° C. Samples were analyzed by flow cytometry.
Soluble DC-SIGN-Fc Adhesion Assay.
The soluble DC-SIGN adhesion assay was performed by ELISA42. Antigens (either 3.75×106 bacterial cells/well or 5 μg/ml, in case of LPS or glycoconjugates) were coated in ELISA plates and soluble DC-SIGN-Fc (1 □g/ml in TSM) binding was determined by an anti-humanIg-Fc ELISA. When indicated, DC-SIGN-Fc was preincubated with with 20 ug/ml mAb, 200 ug/ml mannan or 5 mM EDTA for 10 min at room temperature.
Immunohistochemistry
After informed consent by the patients, 2 antral biopsies were collected during gastroscopy. Tissue cryosections were fixed in acetone and incubated with AZN-D1 followed by anti-mouse-HRP. Staining was performed with the ABC-AP Vectastain kit and AEC (Vector Laboratories, Burlingame, Calif.) and sections were counterstained with heamatoxilin. In parallel, the other biopsy was analyzed for H. pylori infection by incubation in urease-medium for 12 hrs. No differences in morphology and staining were observed between non infected and infected individuals.
DC Activation and Th1/Th2 Differentiation
To analyze maturation, DC (180,000 cells) were incubated with H. pylori for 1 hr at multiplicity of infection (M.O.I) of 1, 5, 10, 20, washed and cultured for 20 hrs. Cells were analyzed for maturation markers (CD80, CD86, CD83, HLA-DR) by flowcytometry and supernatant was collected for cytokine ELISA22. For T cell differentiation, DCs were cocultured with H. pylori (M.O.I of 10) for 2 days, washed and incubated with CD45RA+, CD4+T cells (5,000 T cells/20,000 DCs). In parallel, DCs were analyzed for maturation markers, described above, and cytokine production after stimulation with J558 transfected with CD40L kind gift from P. Lane, University of Birmingham, UK) in the presence or absence of IFN-□ (1000 U/ml).
Quiescent T cells were restimulated with PMA (10 ng/ml, Sigma-Aldrich, St. Louis, Mo.) and ionomycin (1 μg/ml, Sigma-Aldrich) for 6 hr, the last 5 hrs in the presence of brefeldin A (10 μg/ml, Sigma-Aldrich). Single-cell production of IL-4 and IFN-γ was determined by intracellular flowcytometry43.
Results
DC-SIGN has been demonstrated to have a carbohydrate specificity for mannose-containing carbohydrates, Lewis antigens and GlcNAc-containing structures (
DC-SIGN is a C-type lectin in particular highly expressed on Dendritic cells. Its function is to recognize antigen and process and present this very efficiently into MHC calss I and II molecules.
It has long been thought that DC-SIGN is a pathogen recognition receptor that recognizes pathogens to activate the immune system, but as it seems now, the long list of pathogens that target DC-SIGN all persist and escape immunity by different mechanisms. We have now four examples of pathogens that target DC-SIGN to survive in the host.
1. HIV-1 and HCV target DC-SIGN to ‘hide’ within DC and escape the intracellular routing to the lysosomal compartment.
2. Secretion of ManLam by mycobacteria targets DC-SIGN to induce downmodulation of TLR induced DC maturation, and induction of IL-10.
3. the human gastric pathogen Helicobacter pylori persists: Lipopolysaccharide phase variants modulate the Th1/Th2 balance through interaction with the dendritic cell lectin DC-SIGN.
4. Lactobacillus target DC-SIGN on DC and induce regulatory T cells, inhibition of recognition of Lactobacillus by DC-SIGN reduces the regulatory state of T cells. It is therefore supposed that DC-SIGN is a receptor that ‘normally’ recognize self-antigen to tolerize (Annual Review Immunology, 2004). We here demonstrate that targeting of DC-SIGN by lactobacilli can indeed induce regulatory T cells. Furthermore it seems that DC-SIGN can also recognize the tumor antigen CEA (CD66e) in particular Lewis X and Y. These tumor antigens may be secreted and suppress DC activation in a similar way as ManLam of mycobacteria tuberculosis.
The interaction of DC with granulocytes is elucidated by the fact that DC-SIGN recognizes CD66a and CD11b on granulocytes. In particular Lewis X antigen on these molecules is recognized. In that way DC-SIGN functions as a cell adhesion receptor that mediates cellular interactions of granulocytes with DC. This cellular adhesive function between granulocytes and dendritic cells has never been reported but it is likely that the granulocyte DC interaction is essential to form a bridge between the innate immune response and the adaptive immune response in such a way that the granulocyte passes the infectious agents to the DC mounting an adequate immune response. In particular the granulocyte activates and matures the DC, inducing upregulation of cytokines and costimulatory molecules which is necessary for DC migration and initiation of the adaptive immune response. The interaction of DC with granulocytes is also seen in vivo in the situation of Crohns disease. A new pathogen that interacts with DC-SIGN on DC is Neisseria Meningitidis. Strain variants demonstrate that in particular one strain mutant IgtB interacts which contains endstending GlcNAc residue. Other experiments (
| Number | Date | Country | Kind |
|---|---|---|---|
| 020796652 | Nov 2002 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/NL03/00781 | 11/7/2003 | WO | 10/5/2005 |
