ImmunoMethods最新文献

Bispecific antibodies (BsAb) are antibodies with two different specificities. BsAb composed of antl-FcγR Ab linked to anti-target Ab have been useful in exploring the function of the three classes of human FcγR. In addition, BsAb have been developed as new agents for immunotherapy which can join together different molecules or cells. In directed or redirected cytotoxicity, BsAb that bind both to target cells (pathogens or tumors) and to triggering molecules on leukocytes such as FcγR are used to focus normal cellular immune defense mechanisms specifically to the tumor cell or infectious agent. Limited clinical trials have demonstrated little toxicity and promising responses. This ability to redirect normal cytotoxic mechanisms to kill tumors, infectious agents, or infected cells makes BsAb powerful new therapeutic tools. In addition, BsAb are being used to target other appropriate molecules to FcγR, including antigens as vaccine adjuvants and immune complexes. This review focuses on BsAb in which one specificity is directed to FcγR on human leukocytes. It considers applications of these reagents and discusses the progress toward an understanding of the construction and use of BsAb in therapy.

The low-affinity Fc receptor for IgE (FcϵRII/CD23) and its soluble form (sCD23, IgE-binding factor) have multiple functions, and enhanced levels of these are associated with various immunological diseases. We established two sensitive ELISA systems using enzyme-conjugated mAb and biotinylated mAb. The detection limits of the ELISA systems were 0.03 and 1.0 ng/ml, which showed good correlation in the range 1.0-10 ng/ml. In the ELISA system using enzyme-conjugated mAb, the average sCD23 concentration in 303 normal healthy volunteers was 1.4 ± 0.3 ng/ml. In the ELISA system using biotinylated mAb, sCD23 levels in normal healthy volunteers showed almost the same values. In patients with autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Sjögren syndrome, progressive systemic sclerosis, and mixed connective tissue disease, the sCD23 levels were significantly higher than those in normal individuals. Furthermore, in Epstein-Barr virus-related disorders after liver transplantation with immunosuppression, plasma levels of sCD23 rapidly Increased to more than 12 ng/ml when clinical symptoms were evident. In addition, the sCD23 values remained high, although elevated GOT levels gradually decreased to standard values and EBV hepatitis improved. These data suggest that sCD23 levels are a sensitive marker of autoimmune diseases and EBV-related disorders in addition to allergic disorders. The ELISA system for sCD23 may be an additional diagnostic tool in estimating the clinical courses of these diseases.

In the past two decades, rapid progress has been made in the field of Fc receptors. One of the reasons for this development has been the advances and improvements in the methodologies used to detect and analyze Fc receptors. This review describes the methods used to detect the murine and human Fc receptors of the various immunoglobulin isotypes at the RNA, protein, and functional level. We have given special attention to the binding assays used to detect Fc receptors through immunoglobulins or anti-Fc receptor monoclonal antibodies as probes. The various Fc receptor types exhibit a marked heterogeneity and we present a list of reagents that can differentiate between the different Fc receptor forms. These methods can detect Fc receptors on the cell membrane, as well as the cytoplasmic and secreted forms. Recent advances in the use of PCR and RNA/DNA analyses to study Fc receptor detection, expression, and function are also reviewed. The advantages and drawbacks of the various experimental approaches are presented. In addition, an appendix provides detailed protocols for the detection of Fc receptors by confocal immunofluorescence microscopy, rosetting, cytofluorometry, immunoprecipitation, and PCR

Murine low-affinity Fc receptors for IgG (FcγRIIbl, FcγRIIb2, and FcγRIII) bind the same IgG subclasses and are not distinguished by available anti-FcγRII/III mAbs (2.4G2). They trigger various biological activities, among which are the internalization of soluble and particulate immune complexes, cell activation, and its regulation. To determine the biological properties of the three murine receptors, each was expressed by stable transfection of corresponding cDNAs in two model cells: the murine lymphoma B cell IIA1.6 and the rat basophilic leukemia cell RBL-2H3. Biological activities of recombinant receptors were triggered with soluble immune complexes or 2.4G2 IgG in IIA1.6 cells, which express no FcγR, and with 2.4G2 Fab or F(ab′)₂, cross-linked with mouse anti-rat F(ab′)₂ in RBL, which express rat FcγR. Conditions for studying cell activation and endocytosis in both cell models are described, as are conditions for studying phagocytosis in RBL cells and antigen presentation or regulation of cell activation in IIA1.6 cells. Internalization of immune complexes was triggered by FcγRIIb2 and FcγRIII, but not by FcγRIIb1. Intracytoplasmic sequences required for phagocytosis and endocytosis could be distinguished in FcγRIIb2, but not in FcγRIII. Cell activation was restricted to FcγRIII. FcγRIII-mediated endocytosis, phagocytosis, and cell activation involved the consensus tyrosine-containing activation motif found in the intracytoplasmic domain of the γ subunit. Regulation of cell activation was induced by both FcγRII isoforms and depended on the same sequence as endocytosis. As a consequence, a single motif can determine more than one biological response of the cell, and a given response may be triggered by several motifs, borne by different FcγR.

The high-affinity receptor f or IgE (FcϵRl) belongs to a class of multimeric receptors associated with nonreceptor tyrosine kinases. It has been assumed that FcϵRI β and γ chains, which have extensive cytoplasmic domains, play an important, although undefined role in coupling the receptor to signal transduction mechanisms. The results reviewed here suggest a synergistic effect of these two chains in the initiation of FcϵRI signaling. According to our model, receptor engagement can activate kinase(s), such as lyn, already bound to the receptor under resting conditions. The receptor phosphorylation following this activation can be responsible for recruitment and activation of other signaling molecules, such as syk, which can then activate downstream effector molecules. This model could be extended to include other multimeric receptors, such as the T- and B-cell receptors and the low-affinity receptor for IgG (FcγRIII), that control the activation of cytoplasmic tyrosine kinases.

The low-affinity receptor for IgG, FcγRll, and the high-affinity receptor for IgE, FcϵRI, are functionally distinct but structurally homologous receptors. These characteristics have been exploited using a chimeric receptor strategy to examine segments of human FcγRII for IgG-binding function. A series of chimeric receptors was generated by exchanging coding regions of the extracellular ligand-binding regions between FcγRll and the FcϵRI α chain using splice overlap extension by the polymerase chain reaction. The expression of these chimeric receptors in COS-7 cells and analysis of their IgG/IgE binding capacities have enabled the Ig-binding region of FcγRll to be localized to a subregion of the second extracellular domain. The localization of the Ig-binding region of FcγRII has provided the opportunity of performing site-directed mutagenesis to determine the key amino acids involved in the interaction of the receptor with IgG. These findings demonstrate that the chimeric receptor approach is a powerful technique for the dissection of structure/function relationships of structurally related yet functionally different molecules.

The complex family of human IgG Fc receptors show a wide cellular distribution and a strong functional heterogeneity. To date, eight different genes that are transcribed into at least 12 different mRNAs have been recognized. Although corresponding products have been identified for only some of the transcripts, in vivo at least six different FcγR isoforms are shown to be present on the surface of all kinds of leukocytes. Upon interaction with immunoglobulin, FcγR mediate a variety of biological responses such as phagocytosis, endocytosis, release of inflammatory mediators, and antibody-dependent cellular cytotoxicity. One of the main questions in FcγR research is determining the specific relationship between a particular FcγR isoform and a particular effector mechanism. The availability of the cDNA and genomic clones corresponding to the different isoforms, combined with different gene transfer systems in vitro and in vivo and the recently developed procedure of gene knockout by homologous recombination, allows the generation of stably transfected cell lines and transgenic animals that express one particular FcγR isoform. These model systems enable us to study the function of a particular FcγR isoform in the context of a particular cell type in vitro and in the context of the intact immune system in vivo.

When studying the interaction between two molecules, one needs to describe the interface to compare and contrast it with other systems. Three geometric methods for analyzing these interactions have been developed: (i) calculation of pairwise atomic contacts; (ii) calculation of surface area; (iii) calculation of packing volume. This paper describes these methods and focuses on the first two to describe antibody-antigen interactions. Application of these methods reveals some ways in which antibody interactions differ with different size antigens.

Although many antibodies have been crystallized, the number of structures determined in both their complexed and unliganded forms remains relatively small. With the recent improvements in the use of molecular replacement (MR), the structure determination of Fabs and Fab-complex structures can proceed more rapidly, but crystallization often remains a major obstacle. Substantial improvements in methodologies have helped with the success rate in the crystallizations of Fabs and Fab-antigen complexes that are beyond previous expectations. Crystallization and structure determination have been directed mainly toward Fab fragments. The reason for this choice remains linked both to the ease with which the structure of Fabs can be determined and to the difficulties that have been presented by the crystallization of whole immunoglobulins. Such difficulty is currently believed to be due to flexibility or conformational heterogeneity of the IgG as well as the added heterogeneity from the glycosylation of the Fc fragment. Fabs share some of the same problems mainly because of the degree of heterogeneity that is the result of the proteolytic cleavage used to fragment the immunoglobulins, of the flexibility in elbow regions, and in some cases of glycosylatlon. A systematic approach to the cleavage, purification, and analysis of the resultant product can yield inmunoglobulin fragments amenable to crystallization. A rational screening of crystallization conditions with extensive use of seeding can In most cases enable progress from small microcrystalline aggregates to large X-ray-quality crystals. Such methodologies have become so effective that Fabs are now being used as tools to aid in the crystallization of other molecules that have been found difficult to crystallize by themselves.