Ig Molecules Research Articles

Multiple B29 containing complexes on murine B lymphocytes. Common and stage-restricted Ig-associated polypeptide chains.

The B29 gene is specifically expressed in all cells of the B lymphocyte lineage, and the B29 protein is disulfide-linked to the protein product of at least one other gene, known as mb-1. The noncovalent association of these heterodimers with Ig H chains is thought to be required for surface expression and signal transmission by B cell Ag receptors. We now demonstrate by two-color immunofluorescence a direct correlation between B29 density and surface Ig expression on normal spleen and bone marrow cells. The proportion of B29 in Ag receptor complexes appears to be relatively constant across major B lymphocyte subpopulations. Multiple B29-containing heterodimers were resolved on normal spleen cells by surface labeling, immunoprecipitation, two-dimensional gel analysis, and immunoblotting. As with lymphoma cells in our earlier study, the conditions of detergent extraction were critical to detection of certain species. Many laboratories have observed a family of 69- to 85-kDa heterodimers that are extracted with digitonin. These species are clearly Ig-associated, and are coprecipitated with anti-Ig antibodies. We found that extraction with Triton X-100 revealed an additional pair of 52- to 58-kDa heterodimers, where B29 was disulfide-bonded to a protein of approximately 23 kDa. The latter was detectable by immunoblotting with antibodies to extracellular, but not cytoplasmic, portions of mb-1. We found that, with mature cells, both conventional and low molecular mass heterodimers were solubilized with digitonin, but only detectable if Triton was present during immunoprecipitation. Thus, a protein having partial serologic identity with mb-1 forms heterodimers that are cryptic on splenic B cells, and possibly not directly associated with surface Ig molecules. In contrast, both types of heterodimers were readily detectable on late stage pre-B cells, regardless of detergent used for extraction or antibody used for immunoprecipitation. In that situation, both low- and high molecular mass heterodimers were associated with surface Ig. These findings increase our understanding of the B lymphocyte Ag receptor complex and indicate that its components may change as a function of differentiation.

Changes in the Expression of Ig‐Associated Proteins on B Lymphocytes Activated by Anti‐IgM Antibodies

Binding of antigen to receptor complexes on B cells elicits a cascade of intracellular signalling events leading to proliferation and, together with T-cell help, Ig secretion. Components of the antigen receptor (AgR) complex have been demonstrated to be either covalently bound or associated with surface Ig (sIg) molecules. The function of these proteins is still unknown. In order to address this question, we have stimulated B cells with anti-mu antibodies and have studied possible changes in the expression of AgR complexes. After anti-mu stimulation, the IgM molecules disappeared rapidly from the cell surface together with the covalently bound proteins. The IgM molecules were internalized and probably degraded. The IgM-associated heterodimer Ig-alpha/Ig-beta was also removed from the cells, leaving the IgD-associated heterodimer unaffected. Two proteins showed an enhanced association with sIg after 15 min and then were gradually removed from the cell surface. Two other proteins became increasingly attached to sIg. This association remained stable for the rest of the culture period (up to 4 h). Further studies are underway to characterize these proteins more closely and to examine possible interactions with downstream members of the signalling cascade.

Antibodies to Trypanosoma cruzi express idiotypic patterns that can differentiate between patients with asymptomatic or severe Chagas' disease.

Immunization of rabbits with pools of immunoaffinity-purified anti-Trypanosoma cruzi epimastigote antibodies derived from patients with different clinical forms of Chagas' disease induces antiidiotypic sera that can distinguish between anti-epimastigote antibodies from patients with asymptomatic (indeterminate (IND)) or severe (cardiac (CARD)) Chagas' disease. These idiotypically different anti-EPI antibodies from patients with the different clinical forms do not differ in their anti-epimastigote activities or isotypes. Analysis of immunoaffinity purified antibodies from individual chagasic patients by specific competitive ELISA generally confirms that Id-specific rabbit antisera can differentiate the clinical forms of the source of the antibodies. Based on these data, immunoaffinity-purified antibodies from patients share many Id with those from IND patients, although antibodies from IND patients express much lower levels of the distinctive Id characteristic of CARD patients. Reduction and alkylation of antibodies from IND patients reduces somewhat, but does not abolish, the ability of their Id to be recognized idiotypically, and to effectively inhibit in competitive ELISA. In contrast, reduction and alkylation of antibodies from CARD patients almost completely eliminates the ability of their predominant Id to be either recognized by, or inhibit, the appropriate systems. These data imply that the expression of the major Id that define CARD patients by these serologic anti-Id systems is largely dependent on the tertiary conformation of the Ig molecule. This agrees with our earlier studies on the respective differential abilities of CARD vs IND Id to stimulate anti-Id T cells by direct stimulation vs processing and presentation mechanisms.

Interactions of cell-surface galactosyltransferase with immunoglobulins

Detection of the activity of β-1,4-galactosyltransferase (β-1,4-GT) in suspensions of viable mouse hepatocytes, the human hepatoma cell line Hep G2, the human colonic adenocarcinoma cell line HT-29, the monocyte-like cell line U937, and human splenic B and T lymphocytes suggested the presence of β-1,4-GT, in an enzymatically active form, on plasma membranes. The presence of β-1,4-GT on cell surfaces was also indicated from the effect of trypsinization of live cells, which significantly reduced cell surface β-1,4-GT activity, but did not affect the activity associated with cytoplasmic membranes. Furthermore, the presence of β-1,4-GT on the cell surface was demonstrated by indirect immunofluorescence staining of cells with anti-β-1,4-GT antibody. The detection of radioactivity in immunoglobulins (Ig) and their component chains after incubation with suspensions of intact cells in the presence of Mn 2+ and UDP-[ 3H]-galactose, indicated that Ig molecules were galactosylated. In the absence of UDP-[ 3H]-galactose, β-1,4-GT on cell surfaces, or immobilized on Sepharose-4B, formed stable complexes with galactose acceptors, including Ig. The efficiency of binding decreased in the order: J chain > α chain > μ chain > polymeric IgA2 > monomeric/polymeric IgA1 > IgM > IgG. Thus, β-1,4-GT could act as a cell-surface receptor for Ig through a cation-dependent, lectin-like association of the β-1,4-GT with the carbohydrate moieties of the Ig. This was confirmed by indirect surface immunofluorescence and radiolabeled ligand binding assays. The binding was inhibitable by EDTA, α-lactalbumin (in the presence of glucose), GlcNAc, or uridine 3',5'dialdehyde. At 37°C, the apparent affinity constants and association rate constants of interaction between cell surface β-1,4-GT on glutaraldehyde-fixed HT-29 and U937 cells and α2 chain or monomeric IgA1 were in the range from 7.1 × 10 7 to 4.6 × 10 8 M −1 and from 1 × 10 5 to 3 × 10 6 M −1 s −1, respectively. The dissociation rate constants and half time of dissociation calculated from these data were in the range from 2.1 × 10 −2 to 5.0 × 10 −4 s −1 and from 33 to 1380 s, respectively. The number of α2 or IgA1 molecules bound per HT-29 and U937 cell were in the range from 1.9 × 10 5 to 1.3 × 10 6. The binding of IgA by the cell surface β-1,4-GT was not associated with internalization or the catabolic degradation of the ligand.

The T cell receptor αβ V‐J shuffling shows lack of autonomy between the combining site and the constant domain of the receptor chains

In order to assess the structural independence of the T cell receptor (TCR) combining site from the rest of the molecule we have generated two recombinant chains consisting of a TCR V-J alpha region linked to the C beta and a TCR V-J beta linked to the C alpha. If the V and C domains of the TCR form independent domains, as has been shown for the Ig molecules, we would expect to obtain a functional chimeric TCR. Interestingly, it was found that the shuffled molecules are produced intracellularly in T cell hybridomas, but are not expressed on the cell surface. To explain this failure of the shuffled molecules we propose that the TCR has a more compact structure, compared to the Ig, and that it is indispensable to keep a longitudinal inter-domain contact between the V-J and C portion to have a functional molecule.

The human Ig-beta cDNA sequence, a homologue of murine B29, is identical in B cell and plasma cell lines producing all the human Ig isotypes.

The B cell Ag receptor complex consists of at least two disulfide-linked, heterodimeric structures: the clonally restricted membrane Ig (mIg) molecule and the nonpolymorphic Ig-alpha:Ig-beta protein dimer. The latter molecule is encoded by two separate genes, mb-1 and B29. The DNA sequences of murine and human mb-1 and murine B29 have been determined previously. This study describes the sequence of the full-length human cDNA homologue of the murine Ig-beta/B29 message. The human sequence codes for a protein that displays the typical subunit features of a transmembrane member of the Ig superfamily. The transmembrane and intracytoplasmic domains exhibit striking nucleotide and amino acid sequence similarity between the two species. These regions show almost complete conservation of areas presumed to be involved in noncovalent interactions with other members of the receptor complex and with intracellular kinases and cytoskeletal components. The only sequence dissimilarity seen in these presumed critical areas involves the Y-E-G-L-N motif, a potential target for tyrosine phosphorylation. In contrast, the extracellular portion is much more divergent. Inasmuch as similar patterns of species diversity have been reported for Ig-alpha, the Ig-alpha and Ig-beta molecules may have coevolved to maintain species-specific extracellular interactions between one another and with mIg. Similar to the Ig-alpha molecule, the Ig-beta sequence is identical in B lineage cells expressing all five Ig isotypes. However, in contrast to the Ig-alpha molecule, the Ig-beta sequence is expressed at apparently similar levels in terminally differentiated, mIg- plasma cells as well as in mIg+, mature B cells. These data suggest that Ig-beta has functions in addition to those associated with surface mIg expression.

Presentation of a viral T cell epitope expressed in the CDR3 region of a self immunoglobulin molecule.

Synthetic peptides corresponding to microbial epitopes stimulate T cell immunity but their immunogenicity is poor and their half-lives are short. A viral epitope inserted into the complementarity-determining region 3 (CDR3) loop of the heavy chain of a self immunoglobulin (Ig) molecule was generated from the Ig context and was presented by I-Ed class II molecules to virus-specific, CD4+ T cells. Chimeric Ig-peptide was presented 100 to 1000 times more efficiently than free synthetic peptide and was able to prime virus-specific T cells in vivo. These features suggest that antigenized Ig can provide an improved and safe vaccine for the presentation of microbial and other peptides.

FcγR-expressing monocytes are necessary for PWM-induced proliferation of Saimiri sciureus peripheral blood mononuclear cells

When used as a polyclonal activator of blood immunocompetent cells in the squirrel monkey, pokeweed mitogen (PWM) failed to induce lymphoproliferation unless either autologous or human serum was added to the culture medium, instead of a serum assumed to be deprived of Ig such as foetal bovine serum. We thus attempted to delineate activation processes initiated by PWM and which required both monocytes and native Ig molecules. For instance, the physical presence of live monocytes within lymphocyte suspensions vs. paraformaldehyde-fixed monocytes or products of monocytic origin was an absolute prerequisite for inducing squirrel monkey lymphocytes to proliferate in the presence of PWM. In addition, a combination of monocytes and native Ig were further shown to induce lymphocytes to proliferate in the presence of PWM. We therefore questioned whether Fc gamma R on the surface of monocytes vs. Fc gamma R expressed by other cells were involved in the initiation of PWM-driven lymphoproliferation. PWM may represent a useful tool for studies of Fc gamma R-mediated immunological processes.

Engineered immunoglobulin molecules as vehicles for T cell epitopes.

The variable regions (V) of immunoglobulins (Ig) bear antigenic determinants that can stimulate both humoral and cellular immune responses subsequent to hetero, allo or iso-immunization. The expression of these determinants by Igs usually correlates with the presence of specific amino acid residues within the CDR loops of the V regions. Since the CDR loops varies in length, we reasoned that they would represent permissive sites to insert foreign peptides and create antigenized Igs expressing selected determinants. Taking advantage of these properties and the fact that Igs are self and long-lived molecules, we expressed a CTL and a helper epitope of influenza virus nucleoprotein and hemagglutinin respectively, within the heavy chain CDR3 loop of an anti-arsonate antibody. We found that foreign peptides comprised of 11 to 15 amino acid residues can be expressed within the V region of the heavy chain without alteration of pairing with the light chain. More striking, the cellular processing machinery is able to generate the peptides from the Ig context which were then recognized by specific T cells. Furthermore, the engineered Igs are able to induce T cell responses specific for the inserted peptide and for influenza virus. The use of engineered Ig molecules as vehicles for T and B cell peptides might represent a valuable strategy to generate safe, long lived reagents able to stimulate strong specific immune responses. This would then overcome the short half life of synthetic peptides based vaccines and the side effects seen in case of recombinant viral proteins or inactivated viruses based vaccines.

Anti-peptide reagent identifies a primary-structure-dependent, cross-reactive idiotype expressed on heavy and light chains from a murine monoclonal anti-cd4

A synthetic peptide corresponding to the second complementarity determining region (CDR2) of the immunoglobulin (Ig) variable (V) region heavy (H) chain (CDR2V H) of anti-Leu3a, a murine monoclonal antibody specific for the human CD4 molecule, was used to elicit the production of specific rabbit anti-peptide antibodies. The rabbit anti-peptide antiserum was tested for reactivity against the immunizing peptide, anti-Leu3a, and a panel of mouse monoclonal anti-CD4. Only the immunizing peptide and anti-Leu3a were recognized by ELISA, whereas the H chains of anti-Leu3a and five other monoclonal anti-CD4 preparations were recognized by Western blot analysis. These data suggest that linear structures corresponding to the CDR2V H are not normally exposed on the surface of these monoclonal antibodies and become accessible only upon unfolding of the Ig molecule. In addition, Western blot analysis demonstrated that the anti-CDR2V H peptide antiserum was able to recognize the Ig light (L) chain of anti-Leu3a. This reactivity to both H and L chains from anti-Leu3a was ascribed to a homologous five amino acid sequence region shared by the two chains. The region of homology was associated with the third framework (FR3) of the L chain and was inclued as a portion of the sequence in the CDR2V H synthetic peptide. This observation was confirmed by the ability of the CDR2V H anti-peptide antiserum to bind the L chains of three mouse myeloma proteins that exhibited the five amino acid sequence region of homology within their respective FR3. Together, these data provide information on the structural basis of idiotypes shared by the H and L chains from the same antibody molecule and indicate that five amino acids might be sufficient to define a minimal continuous idiotypic determinant.

Chimeric T cell receptor-immunoglobulin molecules: function and applications.

The antigen-specific receptors on T and B cells are related by sequence similarities, mechanisms for the generation of diversity, and a common protein domain structure. In contrast, the form of antigen recognition for T- and B-cell antigen receptors is entirely different. Whereas the B cell antigen receptor, i.e., membrane-bound immunoglobulin (Ig), has the potential to recognize a vast diversity of chemical determinants, the T cell antigen receptor (TCR) invariably recognizes oligomeric peptides bound to major histocompatibility complex molecules. A question is whether the variable domains of the TCR and Ig are similar in structure, and if so, can they be substituted one for the other. Recent experiments show that, in some combinations, the variable region of Ig can substitute for the variable region of a TCR, and convey, to a reactive T lymphocyte, the antigen specificity of an Ig molecule. This type of receptor engineering may have interesting applications in disease therapy.

Targeting of [ 111In]biocytin to cultured ovarian adenocarcinoma cells using covalent monoclonal antibody—Streptavidin conjugates

Three monoclonal antibodies (mAb) directed against the human ovarian adenocarcinoma cell line HEY, were substituted with maleimide and covalently bonded to thiolated streptavidin. The conjugates were separated from unreacted reagents by successive affinity chromatography on protein A—Sepharose and iminobiotin columns. Purified conjugates consisted of an immunoglobulin (Ig) monomer bound to a streptavidin tetramer through a covalent bond between the Ig molecule and one of the streptavidin subunits. The conjugates were able to specifically target [ 111In]biocytin to HEY cells in vitro in the presence of human serum and ascitic fluid from ovarian cancer patients.

Membrane region of surface IgM is not sufficient for transducing growth inhibitory signals in an immature B cell line WEHI-231.

The murine B lymphoma line WEHI-231 is representative of immature B cells. Like normal immature B cells, WEHI-231 is susceptible to growth arrest following cross-linking of surface IgM (sIgM). Previously, we have shown using a WEHI-231 immunoglobulin (Ig) delta-transfectant that sIgD cross-linking failed to initiate growth arrest, in contrast to sIgM. In this report, we extend our research to investigate the structural requirement of Ig mu chain for regulating growth inhibition. Recombinant, chimeric Ig molecules delta/mu m and mu/delta m consisting of exons encoding extracellular delta and mu domains and membrane regions of different isotypes were constructed and introduced into WEHI-231 cells. A similar approach was used for sIgG2b-expressing transfectants. Our findings indicate that the mu m region is not sufficient for regulation of growth inhibition in WEHI-231 cells and suggest that additional extracellular region(s) of mu chain may be required for this response.

The cytotoxic action of lymphokine activated killer cells upon the human glioma cell line U251 is stimulated by bispecific monoclonal antibody (MoAb) constructs.

The ability of IL-2 stimulated mononuclear cells to kill the human glioblastoma cell line U251 has been investigated. Highest cytotoxic activity was generated in low cell density cultures incubated for 15 days with 250-1000 U/ml IL-2. Sub-optimal killing was noted, with cells only exposed to IL-2 for three days. Under the latter conditions, bispecific monoclonal antibodies (MoAbs) of either anti-CD3 or anti-CD16 and an anti-NCAM MoAb stimulated LAK cell activity markedly. Anti-CD16 conjugates were found more effective than anti-CD3 and (Fab')2 constructs more efficacious than those made with whole Ig molecules. Maximal stimulation of LAK cell activity was noted with bispecific MoAbs. Little effect was observed with either single or mixtures of monomeric MoAbs. Furthermore, no effect of bispecific MoAbs was observed when target cells lacked expression of NCAM. These results could be of clinical importance as it is not always feasible to screen LAK cells for optimal activity before administration to patients. Whilst bispecific MoAbs have no effect on optimally stimulated LAK cells, they are not inhibitory and can stimulate killing under sub-optimal IL-2 stimulation.

Membrane IgM-mediated signaling of human B cells. Effect of increased ligand binding site valency on the affinity and concentration requirements for inducing diverse stages of activation.

The potential for ligand-initiated signal transduction through B cell membrane IgM is assessed in terms of ligand concentration, binding site valency, and binding site affinity for membrane Ig. Estimates of the physicochemical requirements for achieving G0* enhancement of class II MHC expression, G1 entry, and S phase entry in human B cells were made by comparing the stimulatory effects of three affinity-diverse anti-Cmu2 mAb when in bivalent (unconjugated) form, or as mAb-dextran conjugates with low binding site valency (oligovalent ligands) or high binding site valency (multivalent ligands). An increase in binding site number (and concomitant molecular mass) caused a profound reduction in both the minimal concentration and affinity requisites for B cell activation. The enhancing effect of increased binding site valency was most evident for the signaling of those most distal stages in B cell activation, i.e., G1 and S phase, which were difficult to induce with bivalent ligands. The results suggest that highly multimeric TI-2 Ag may be good immunogens because they are able to elicit a full activation response not only from infrequent high affinity B cells, but also from a substantial proportion of the many lower affinity Ag-specific B cells in virgin B cell populations. Interestingly, the activation of B cells by ligands with binding sites of high intrinsic affinity (Ka = 5 x 10(8) M-1) was less influenced by increases in binding site valency than was B cell activation by ligands with intermediate binding site affinity (Ka = 2 x 10(7) M-1). This suggests that the minimal epitope valency requirement for T cell-independent B cell activation by mIg cross-linking Ag may be dependent on the intrinsic affinity with which membrane Ig molecules on a given B cell interact with the redundantly expressed epitopes.

Cells expressing an H chain Ig gene carrying a viral T cell epitope are lysed by specific cytolytic T cells.

The epitope corresponding to amino acid residues 147-161 of the nucleoprotein (NP) of influenza A virus is recognized by CTL in association with H-2Kd class I Ag. Herein, we engineered an Ig molecule carrying this CTL epitope by replacing the diversity gene segment of the H chain V region of an anti-arsonate antibody with an oligonucleotide that encodes the CTL epitope. The chimeric H chain gene was expressed either alone or together with the parental L chain in the nonsecreting BALB/c myeloma B cell line, SP2/0. The Ig produced by cells transfected with both the chimeric H chain and parental L chains genes expressed the NP epitope but lost the original arsonate binding activity. In addition, SP2/0 cells expressing the chimeric H chain either alone or together with the parental L chain were lysed by class I restricted NP-epitope specific CTL. By contrast, SP2/0 cells pulsed with soluble chimeric Ig molecules were not lysed by the specific CTL. These observations indicate that: 1) this particular CTL epitope can be expressed on Ig molecules without altering the H and L chain pairing; 2) this CTL epitope can be generated from this chimeric Ig in which it is surrounded by flanking regions distinct from those of the viral NP; and 3) the generation of this CTL epitope from the Ig molecule requires the endogenous pathway as do viral proteins.

Photolithographic Immobilization of Biopolymers on Solid Supports

The new technique of site-specific immobilization of biomolecules, for example antibodies, may contribute to the development of biosensors and ligand-binding assays. (The key step is shown schematically here.) A benzophenone-derivatized surface is treated with a solution of the antibody (Ig) and then irradiated through a mask with a checkerboard pattern of transparent and opaque fields. The Ig molecules are bound covalently only in areas which were exposed to light. This was proved by epifluorescence microscopy after the surface was washed free of unbound Ig.

Identification of a Receptor on the Vitelline Membrane of Bovine Oocytes that Recognizes Bovine Immunoglobulin G

In order to investigate whether Ig molecules affect bovine fertility, bovine follicular oocytes were tested for an oolemmal receptor that recognized IgG. The zona pellucida was removed from 296 oocytes. The zona-free oocytes were then incubated with purified bovine IgG, its Fc fragment, or its Fab fragment. They were subsequently washed and incubated with the appropriate rabbit anti-bovine IgG (whole IgG-specific, Fc-specific, or Fab-specific), which was covalently attached to polyacrylamide beads (immunobeads). Bead attachment was scored using light microscopy. Bovine IgG and bovine IgG-Fc bound to the oocytes, whereas the IgG-Fab exhibited negligible binding. The data indicate that a bovine oolemmal receptor exists that binds IgG by recognizing the molecule in the region of the Fc-Fab junction. Thus, the binding of IgG or other Ig molecules to bovine oocytes may play a role in sperm and egg interaction.

Evidence for failure of V(D)J recombination in bone marrow pre-B cells from X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) results from a failure of B lymphoid development. We have previously examined pre-B cell hybrids from three patients with XLA and found them to be limited to production of a novel germ line transcript of the Ig H chain locus composed of a leader sequence (LS) spliced to the constant region of mu chain (C mu) as mRNA and polypeptide. These transcripts result from transcriptional activation of the germ line heavy chain locus from an LS exon upstream of the embryonic JH locus. Germ line LS-C mu transcripts are produced by pre-B cells from normal bone marrow and fetal liver, indicating that they are products of normal pre-B cell development, as part of the process of transcriptional activation to provide access for the recombinase. Bone marrow from three patients with XLA has been examined directly by polymerase chain reaction amplification to determine whether the exclusive production of LS-C mu by XLA pre-B cell hybrids is representative of XLA pre-B cells. I report that LS-C mu is the predominant Ig molecule produced by XLA pre-B cells, with limited production of the D mu product of DJH intermediate stage of V(D)J recombination. Mature VHDJH recombinations were not detected with a variety of primers that amplify VH sequences. I conclude that XLA is associated with a limitation in V(D)J recombination that may cause the failure of pre-B cell development.

Construction, purification, and characterization of new interferon gamma (IFN gamma) inhibitor proteins. Three IFN gamma receptor-immunoglobulin hybrid molecules.

Three efficient mouse interferon gamma (MoIFN gamma) inhibitors were constructed, which consist of the MoIFN gamma receptor (MoIFN gamma R) extracellular portion and constant domains of immunoglobulin (Ig) molecules. These are: 1) the constant domain of the mouse kappa chain, 2) the hinge region and the constant domains 2 and 3 of the mouse gamma 2a chain, and 3) the hinge region and the constant domains 2 and 3 of the human gamma 3 chain. The hybrid molecules were expressed in the mouse myeloma cell line J558L and recovered from the supernatants of cell cultures in one purification step. The proteins MoIFN gamma R-M gamma 2a and MoIFN gamma R-H gamma 3 form homodimers, whereas MoIFN gamma R-M kappa is a monomer. All three constructs inhibit the binding of radiolabeled MoIFN gamma to its receptor on L1210 cells. They are biologically active in vitro, neutralizing the action of MoIFN gamma in an antiviral activity assay. The fusions of Ig regions to the soluble MoIFN gamma R do not decrease the affinity of the binding site for the ligand. MoIFN gamma R-M kappa has about the same affinity as the soluble MoIFN gamma R and the cell surface receptor of L1210 cells in situ, which are also monomers, whereas the dimers MoIFN gamma R-M gamma 2a and MoIFN gamma R-H gamma 3 display a 5-10-fold higher affinity for MoIFN gamma than the monomeric molecules. This is best documented in the efficacy of the inhibitors to antagonize the antiviral activity of MoIFN gamma, as the dimeric constructs are about 10 times more active than MoIFN gamma R-M kappa and the soluble MoIFN gamma R. The hybrid constructs can be used as high efficiency MoIFN gamma inhibitors in mouse models of several pathological states in humans, where IFN gamma is thought to play a disease-promoting role.