Polymorphisms of CD3epsilon in cynomolgus and rhesus monkeys and their relevance to anti-CD3 antibodies and immunotoxins

The monoclonal antibody FN18 has been used as a marker for monkey T cells and as a T-cell-depleting reagent when conjugated to diphtheria toxin that was mutated to prevent binding to non-targeted cells. The antibody recognizes a conformational epitope on the ectodomain of monkey CD3epsilon and displays a range of binding activity to the T cells from different rhesus and cynomolgus monkeys. Our quantitative fluorescence-activated cell sorting analysis of the FN18 reactivity to T cells from different rhesus and cynomolgus monkeys showed that there are at least three levels of FN18 reactivity in the monkeys tested: high, moderate and low. On the basis of available DNA sequence information, we determined the gene structure of rhesus CD3epsilon chain and designed primers that can be used to amplify and quickly sequence the ectodomain of monkey CD3epsilon. Our sequence analysis revealed that the extent of nucleotide sequence variation in this area is greater than that previously reported. In addition to the amino acids at positions 45 and 50, we demonstrated that position 35 of CD3epsilon was also important and substitution of amino acid A for V at this position greatly reduced T-cell reactivity to FN18. We found that T cells from monkeys with high FN18 reactivity all had V, E and R at positions 35, 45 and 50 in CD3epsilon, respectively; those having low FN18 reactivity were homozygous in CD3epsilon with at least one of the changes: V35 to A, E45 to G and R to 50Q, whereas members in the moderate group are heterozygous, having both V and A, E and G, R and Q at these locations. A cytotoxicity assay revealed that T cells from a heterozygous rhesus monkey with moderate FN18 reactivity were much (about 40 times) less sensitive to a FN18-derived immunotoxin than those from a homozygous rhesus monkey having high FN18 reactivity.     

CD3 polymorphism in cynomolgus monkeys (Macaca fascicularis)

Cynomolgus monkeys were divided into two groups in terms of the reactivity of their lymphocytes with the FN18 monoclonal antibody, which is directed to the CD3 of rhesus monkeys. It was shown that 24 (12.2%) out of 196 monkeys did not have lymphocytes that reacted with the FN18, although T cells from those animals responded well to mitogenic stimulation. We have determined the nucleotide sequences of the CD3delta, CD3gamma, and CD3epsilon chains and found that two amino acids of the CD3epsilon chain of the FN18 non-reactive monkeys were different when compared with the FN18 reactive monkeys. Our results indicated that the CD3epsilon molecule of cynomolgus monkeys is polymorphic at the epitope level, which is recognized by the FN18 monoclonal antibody.     

Detection of CD3epsilon polymorphism in cynomolgus monkeys by a method based on RFLP

We previously reported that peripheral lymphocytes from about 12% of cynomolgus monkeys lacked reactivity with anti-rhesus monkey CD3 monoclonal antibody (FN18). The nucleotide sequence analysis of the genes encoding CD3 component proteins revealed that a single amino acid substitutions found in the CD3epsilon chain determined the phenotype. In this study, we attempted to develop a method based on the restriction fragment length polymorphism (RFLP) and apply it for determination of the genotypes of individual monkeys. Comparison of the phenotype determined by fluorescence-activated cell sorter analysis with the genotype determined by RFLP analysis revealed that the FN18 -positive trait was dominant over the FN18-negative trait. It was also revealed that allele frequency was significantly different among macaques depending on the geographical region where their ancestors were derived from.     

A monoclonal anti-IgE antibody against an epitope (amino acids 367-376) in the CH3 domain inhibits IgE binding to the low affinity IgE receptor (CD23)

We have produced three different mAb specific for human IgE-Fc. Their binding pattern to either heat-denatured IgE or a family of overlapping IgE-derived recombinant peptides and their ability to affect interaction of IgE with its low affinity receptor Fc epsilon R2/CD23 demonstrate that they recognize distinct epitopes on the IgE molecule. All three mAb were able to induce basophil degranulation as measured by the induction of histamine release. mAb 173 recognizes a thermolabile epitope in the CH4 domain. It does not affect the binding of IgE to Fc epsilon R2/CD23. mAb 272 recognizes a thermostable epitope that maps to a sequence of 36 amino acids (AA) spanning part of the CH2 and CH3 domain and it does not affect the binding of IgE to Fc epsilon R2/CD23. mAb 27 recognizes a thermolabile epitope located on a 10 AA stretch (AA 367-376) in the CH3 domain. This area contains one N-linked oligosaccharide (Asn-371), but the antibody is not directed against carbohydrate because it binds to Escherichia coli-derived IgE peptides. mAb 27 inhibits the binding of IgE to Fc epsilon R2/CD23 but is still capable of reacting with IgE already bound to Fc epsilon R2/CD23. These data suggest that upon binding to Fc epsilon R2/CD23, the IgE molecule engages one of two equivalent-binding sites close to the glycosylated area of the CH3 domain.     

MAb 18D3 Triggering of Integrin β1 Will Prevent but Not Terminate Proliferation of Human T Cells

Triggering of integrins can deliver signals that will regulate T cell activation and proliferation when coupled with TCR/CD3 signaling. While co-activation stimuli can be achieved either with immobilized natural ligands or immobilized monoclonal antibodies specific for various integrin subunits, counterposing effects can be delivered by ligation of the integrin β1 chain (CD29) resulting in the downregulation of T cell proliferation. Thus, integrins may play a pivotal role in cell activation and are involved in both positive and negative regulatory pathways. In this report, anti-β1 mAb 18D3 was used to investigate the role of β1 in the negative regulation of T cell proliferation. T lymphocytes were stimulated to proliferate when activated with immobilized mAb to CD3 in conjunction with all of a panel of immobilized mAb to different α4 (CD49d) and β1 epitopes, except the anti-β1.1 mAb 18D3. In soluble form, mAb 18D3 inhibited the induction of DNA synthesis dependent on costimulation of CD3 and the integrin α4 subunit by a mechanism independent of anti-adhesive properties. In kinetic experiments, the addition of mAb 18D3 effectively inhibited the ultimate induction of DNA synthesis at all time points until the time coinciding with the onset of T cell proliferation, indicating that triggering the β1.1 epitope may only act to quench activation events prior to cellular commitment to synthesize DNA. MAb 18D3 did not induce cell death nor render cells incompetent for restimulation, but appeared to selectively inhibit IL-2 synthesis with little effect on the induction of IL-2 receptor expression.     

Monoclonal antibodies to murine CD3 epsilon define distinct epitopes, one of which may interact with CD4 during T cell activation

The TCR is comprised of two variable chains that confer specificity, called alpha:beta or gamma:delta, physically associated with five different molecules that comprise the complex known as CD3. Antibodies to this complex are very useful, as they react with all T lymphocytes. A rat mAb to mouse CD3 has been prepared. It reacts with 100% of T cells in all mouse strains tested but with no other cell type. It binds to the CD3 epsilon chain. This antibody activates cloned T cell lines and normal T cells, provided suitable accessory cells and signals are present. This antibody detects a determinant similar to but not identical with those detected by two previously reported hamster anti-CD3 epsilon antibodies. This antibody fixes C efficiently, and it is thus useful for depletion of T cells from bulk populations. Activation of T cells by one of the three different anti-CD3 epsilon antibodies was inhibited by the Fab fragment of anti-CD4, similar to the effects of anti-CD4 Fab on two previously reported anti-TCR V region antibodies that bind a CD3 epsilon-associated epitope. This further defines a site involving TCR V regions and CD3 epsilon with which CD4 appears to associate during T cell activation.     

CD45RA T-cell activation without proliferation by a partial agonist monoclonal antibody to beta1 integrin

CD45RA T cells are fully co-activated by natural beta1 integrin ligands fibronectin (FN) and VCAM-1, as well as monoclonal antibody (mAb) 19H8, which binds a combinatorial epitope of the alpha4beta1 heterodimer. These integrin ligands stimulate CD3-dependent proliferation and the upregulation of early activation markers CD25 and CD69. However, beta1-specific antibody 33B6, which binds to a similar range of the predominant T-cell integrins as natural ligands FN (alpha4beta1 and alpha5beta1) and VCAM-1 (alpha4beta1), failed to costimulate proliferation in the CD45RA subset, while retaining the ability to costimulate early activation markers CD25 and CD69. After addition of exogenous human interleukin-2 to the culture media, 33B6 costimulation of proliferation is restored. These data provide evidence that a branch of the alpha4beta1 integrin-signaling pathway in CD45RA T cells can be independently regulated and exploited through the use of partial agonist ligands, including mAbs to the integrin heterodimer.     

ICAM-3 regulates lymphocyte morphology and integrin-mediated T cell interaction with endothelial cell and extracellular matrix ligands

Leukocyte activation is a complex process that involves multiple cross- regulated cell adhesion events. In this report, we investigated the role of intercellular adhesion molecule-3 (ICAM-3), the third identified ligand for the beta 2 integrin leukocyte function-associated antigen-1 (LFA-1), in the regulation of leukocyte adhesion to ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), and the 38- and 80-kD fragments of fibronectin (FN40 and FN80). The activating anti-ICAM-3 HP2/19, but not other anti-ICAM-3 mAb, was able to enhance T lymphoblast adhesion to these proteins when combined with very low doses of anti-CD3 mAb, which were unable by themselves to induce this phenomenon. In contrast, anti-ICAM-1 mAb did not enhance T cell attachment to these substrata. T cell adhesion to ICAM-1, VCAM-1, FN40, and FN80 was specifically blocked by anti-LFA-1, anti-VLA alpha 4, and anti-VLA alpha 5 mAb, respectively. The activating anti-ICAM-3 HP2/19 was also able to specifically enhance the VLA-4- and VLA-5-mediated binding of leukemic T Jurkat cells to VCAM-1, FN40, and FN80, even in the absence of cooccupancy of the CD3-TcR complex. We also studied the localization of ICAM-3, LFA-1, and the VLA beta 1 integrin, by immunofluorescence microscopy, on cells interacting with ICAM-1, VCAM-1 and FN80. We found that the anti-ICAM-3 HP2/19 mAb specifically promoted a dramatic change on the morphology of T lymphoblasts when these cells were allowed to interact with those adhesion ligands. Under these conditions, it was observed that a large cell contact area from which an uropod-like structure (heading uropod) was projected toward the outer milieu. However, when T blasts were stimulated with other adhesion promoting agents as the activating anti-VLA beta 1 TS2/16 mAb or phorbol esters, this structure was not detected. The anti-ICAM-3 TP1/24 mAb was also unable to induce this phenomenon. Notably, a striking cell redistribution of ICAM-3 was induced specifically by the HP2/19 mAb, but not by the other anti-ICAM-3 mAb or the other adhesion promoting agents. Thus, ICAM-3 was almost exclusively concentrated in the most distal portion of the heading uropod whereas either LFA-1 or the VLA beta 1 integrin were uniformly distributed all over the large contact area. Moreover, this phenomenon was also observed when T cells were specifically stimulated with the HP2/19 mAb to interact with TNF alpha-activated endothelial cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

A novel LFA-1 activation epitope maps to the I domain

A panel of 21 alpha-subunit (CD11a) and 10 beta-subunit (CD18) anti-LFA- 1 mAbs was screened for ability to activate LFA-1. A single anti-CD11a mAb, MEM-83, was identified which was able to directly induce the binding of T cells to purified ICAM-1 immobilized on plastic. This ICAM- 1 binding could be achieved by monovalent Fab fragments of mAb MEM-83 at concentrations equivalent to whole antibody, was associated with appearance of the "activation reporter" epitope detected by mAb 24, and was completely inhibited by anti-ICAM-1 and LFA-1 blocking mAbs. The epitope recognized by mAb MEM-83 was distinct from that recognized by mAb NKI-L16, an anti-CD11a mAb previously reported to induce LFA-1 activation, in that it was constitutively present on freshly isolated peripheral blood mononuclear cells and was not divalent cation dependent for expression. The ICAM-1 binding activity induced by mAb MEM-83 was, however, dependent on the presence of Mg2+ divalent cations. Using an in vitro-translated CD11a cDNA deletion series, we have mapped the MEM-83 activation epitope to the "I" domain of the LFA- 1 alpha subunit. These studies have therefore identified a novel LFA-1 activation epitope mapping to the I domain of LFA-1, thereby implicating this domain in the regulation of LFA-1 binding to ICAM-1.     

Isolation, characterization, and expression of cDNA clones encoding the mouse Fc receptor for IgE (Fc epsilon RII)1

We have isolated cDNA clones encoding a mouse low affinity receptor for IgE (Fc epsilon RII) from a cDNA library of BALB/c splenic B cells activated with LPS and IL-4. The 2.2-kb cDNA clone encodes a 331 amino acid membrane glycoprotein that is homologous to human Fc epsilon RII (CD23) and a family of carbohydrate-binding proteins. COS7 cells transfected with the cDNA clones expressed a 45,000 m.w. protein that bound IgE and the anti-Fc epsilon RII mAb, B3B4. Fc epsilon RII mRNA was up-regulated in mouse B cells by culture with IL-4, but not in B cells cultured with IgE. Fc epsilon RII mRNA was detected in IgM+/IgD+ B cell lines, but not in pre-B cell lines or in B cell lines which have undergone differentiation to secrete Ig. The monocyte line P388D1, mast cell lines MC/9 and PT18, and peritoneal macrophages stimulated with IL-4 lacked detectable Fc epsilon RII mRNA, as did Thy-1.2+, CD4+, and CD8+ normal T cells and Thy-1.2+ T cells from Nippostrongylus brasiliensis-infected mice.     

Characterization of the putative avian CD2 homologue.

We describe two mouse mAb recognizing the putative chicken homologue of mammalian CD2 Ag and provide evidence for both structural and functional conservation between the avian and mammalian CD2 molecules. The antibodies were T cell-specific and immunoprecipitated a single diffuse band of Mr 40,000 from lysates of surface-labeled chicken thymocytes and peripheral T cells. Removal of N-linked carbohydrate with endo-beta-N-acetylglucosaminidase F revealed the core protein size of Mr 25,000. A rabbit antiserum raised against a synthetic peptide (CD2-300), composed of 18 amino acid residues of the conserved cytoplasmic domain of human, mouse, and rat CD2, precipitated an Ag similar to chicken CD2. Sequential precipitation with CD2-300 antiserum indicated the conservation of an avian and mammalian CD2 epitope. CD2 expression on thymocytes starts at day 11 of embryonic development, and, during subsequent development, thymic gamma delta cells are all CD2+, whereas most peripheral gamma delta-T cells lack CD2. Functional conservation between the chicken and mammalian CD2 molecules was demonstrated by the induction of DNA synthesis in chicken thymocytes and peripheral T cells with the combination of anti-CD2 mAb and PMA.     

Differential regulation of the low affinity Fc receptor for IgE (Fc epsilon R2/CD23) and the IL-2 receptor (Tac/p55) on eosinophilic leukemia cell line (EoL-1 and EoL-3)

Two types of activation Ag, low affinity FcR for IgE (Fc epsilon R2)/CD23 and IL-2R (Tac/p55), were expressed and differently regulated on human eosinophilic leukemia cell lines (EoL-1 and EoL-3). Because the binding of IgE on EoL-3 cells was completely inhibited by H107 (anti-Fc epsilon R2/CD23 mAb) but not by irrelevant mAb, essentially all the low affinity Fc epsilon R2 on EoL-3 seemed to be the Fc epsilon R2/CD23 molecules. Both IL-4 and IFN-gamma enhanced the surface expression of Fc epsilon R2, whereas IL-1, IL-2, and IL-5 showed no effects, as determined by surface staining with anti-Fc epsilon R2 antibody (H107). In contrast to Fc epsilon R2 up-regulation, IL-4 and IFN-gamma showed a differential effect on the regulation of IL-2R (Tac/p55). Whereas IFN-gamma up-regulated the receptor expression of IL-2R/Tac, IL-4 did not. The result suggests that these lymphokines are involved in the different aspects of the activation pathway of the eosinophils. The possible role of Fc epsilon R2 and IL-2R on the function of eosinophils in allergic reaction is discussed.     

Analysis of a human sperm CD52 glycoform in primates: identification of an animal model for immunocontraceptive vaccine development

Sperm agglutination antigen-1 (SAGA-1) is a human male reproductive tract glycoform of CD52. Unique modification of CD52 N-linked oligosaccharide chains in the epididymis and vas deferens results in the appearance of a carbohydrate epitope that is localized over the entire surface of human spermatozoa. SAGA-1 was characterized by the sperm-inhibitory murine monoclonal antibody (mAb) S19, and it is the target antigen of a human mAb (H6-3C4) associated with antibody-mediated infertility. Collectively, sperm surface localization, antibody inhibition of sperm function, and potential reproductive-tissue specificity identify SAGA-1 as an attractive candidate contraceptive immunogen. To establish an animal model for the study of SAGA-1 in immunologic infertility and immunocontraceptive development, we investigated the appearance of the S19 carbohydrate epitope in nonhuman primates. The S19 mAb demonstrated little to no immunoreactivity by Western blot analysis with protein extracts of spermatozoa from the baboon, marmoset, bonnet, cynomolgus, and pigtailed macaques. Immunohistochemical analysis identified CD52 in the bonnet monkey epididymis; however, the N-linked carbohydrate moiety recognized by the S19 mAb, and unique to SAGA-1, was absent. In contrast, the S19 carbohydrate epitope was identified in chimpanzee sperm extracts by Western blot analysis and in chimpanzee epididymal tissue sections by immunohistochemical analysis, indicating that it is conserved in this close relative of the human. Chimpanzee testis, seminal vesicle, and prostate do not express the S19 epitope. Although anti-CD52 immunoreactivity was identified in the spleen, the carbohydrate moiety recognized by the S19 mAb was absent, corroborating data in the human that demonstrated tissue-specific glycosylation of sperm CD52. Immunofluorescent analysis indicated that the chimpanzee homologue of sperm CD52 was present over the entire spermatozoon. In addition, the S19 mAb agglutinated chimpanzee spermatozoa in a manner similar to the effect observed on human spermatozoa. These data indicate that the distinctive carbohydrate moiety of human sperm CD52 is present in the chimpanzee, and they identify the chimpanzee as the most appropriate primate model to study the potential of this unique CD52 glycoform as a contraceptive immunogen.     

Functional reactivity of WT31 monoclonal antibody with T cell receptor-gamma expressing CD3+4-8- T cells

About 3% of normal peripheral blood T lymphocytes have the phenotype CD3+4-8-. The vast majority of these cells lack the conventional TCR-alpha-, beta complex but express the recently identified TCR-gamma, delta/CD3 receptor complex. These TCR gamma+/CD3+ cells were initially discovered by using as a criterion the lack of reactivity with WT31 mAb. This mAb has been reported to recognize a "framework" epitope on the TCR-alpha, beta/CD3 complex. However, using high concentrations of WT31 mAb, low levels of reactivity with the cell membrane of TCR-gamma+ cells can be observed. This reactivity was significantly increased upon removal of sialic acid residues by neuraminidase. In addition, WT31 mAb is capable to induce lysis by TCR-gamma+ clones. Moreover, immunoprecipitation with WT31 by using cell lysates prepared with the mild detergent digitonin resulted in the isolation of the intact TCR-gamma/CD3 complex. Thus, in contrast to what was previously assumed, WT31 mAb also reacts with a functional epitope present on gamma, delta/CD3 T cells, and therefore lack of reactivity with WT31 mAb is not always a proper hallmark for TCR-gamma-expressing cells.     

Vaccination with allergen-IL-18 fusion DNA protects against, and reverses established, airway hyperreactivity in a murine asthma model

Vaccination with naked DNA encoding a specific allergen has been shown previously to prevent, but not reverse, the development of allergen-induced airway hyperresponsiveness (AHR). To enhance the effectiveness of DNA vaccine therapies and make possible the treatment of established AHR, we developed a DNA vaccination plasmid containing OVA cDNA fused to IL-18 cDNA. Vaccination of naive mice either with this fusion DNA construct or with an OVA cDNA-containing plasmid protected the mice from the subsequent induction of AHR. Protection from AHR correlated with increased IFN-gamma production and reduced OVA-specific IgE production. The protection appeared to be mediated by IFN-gamma and CD8(+) cells because treatment of mice with neutralizing anti-IFN-gamma mAb or with depleting anti-CD8 mAb abolished the protective effect. Moreover, vaccination of mice with preexisting AHR with the OVA-IL-18 fusion DNA, but not with the OVA cDNA plasmid, reversed established AHR, reduced allergen-specific IL-4, and increased allergen-specific IFN-gamma production. Thus, combining IL-18 cDNA with OVA cDNA resulted in a vaccine construct that protected against the development of AHR, and that was unique among cDNA constructs in its capacity to reverse established AHR.     

The inclusion of the type III repeat ED-B in the fibronectin molecule generates conformational modifications that unmask a cryptic sequence.

We have previously reported an anti-fibronectin monoclonal antibody (mAb) (BC-1) which reacts with an ED-B-containing beta-galactosidase-fibronectin fusion protein but not with an identical beta-galactosidase-fibronectin fusion protein in which the ED-B sequence is omitted. In further experiments aimed at localizing more precisely the epitope recognized by this mAb, we demonstrate that 1) the mAb BC-1 is indeed specific for ED-B-containing fibronectin (FN) molecules even though the epitope recognized by this mAb is localized on the type III homology repeat 7 (the one which precedes the ED-B sequence) and 2) in fibronectin molecules lacking the ED-B sequence, this epitope is masked. We further demonstrate that, to mask the epitope recognized by the mAb BC-1, the presence of at least half of the FN type III homology repeat 9 is necessary. We also report the production of the mAb IST-6 which recognizes only FN molecules in which the ED-B sequence is lacking. These data clearly demonstrate that the presence of the ED-B sequence within FN molecules generates conformational modification in the central part of the molecules that unmasks previously cryptic sequences and masks others.     

Costimulation of proliferative responses of resting CD4+ T cells by the interaction of VLA-4 and VLA-5 with fibronectin or VLA-6 with laminin

Given prior evidence that adhesion molecules play critical roles in T cell recognition, it is important to identify new adhesion pathways and explore their role in T cell activation. Our studies of T cell proliferation complement concurrent studies of T cell adhesion; both demonstrate that resting CD4+ human T lymphocytes express the VLA integrins VLA-4, VLA-5, and VLA-6, and can use these receptors to interact with the extracellular matrix (ECM) proteins fibronectin (VLA-4 and VLA-5) and laminin (VLA-6). VLA-dependent interaction of resting human CD4+ T cells with fibronectin (FN) and laminin (LN) facilitates CD3-mediated T cell proliferation. Specifically, T cells do not proliferate in response to a wide range of concentrations of a CD3 mAb, OKT3, immobilized on plastic. However, coimmobilization with the CD3 mAb of FN or LN, but not other ECM proteins such as fibrinogen and collagen, consistently results in strong T cell proliferation. mAb blocking studies demonstrate that three VLA integrin receptor/ligand interactions mediate costimulation: VLA-4/FN, VLA-5/FN, and VLA-6/LN. VLA-5-dependent binding to FN but not costimulation by FN can be specifically blocked with peptides containing the RGD (arg-gly-asp) tripeptide sequence whereas VLA-4-dependent binding and costimulation can both be efficiently inhibited by a 12 amino acid peptide, LHGPEILDVPST (leu-his-gly-pro-glu-iso-leu-asp-val-pro-ser-thr), derived from the alternatively spliced IIICS region of FN. The costimulation provided by FN and LN in this system is stronger than and distinct from costimulatory signals provided by cytokines, such as IL-1 beta, IL-6,, and IL-7. These results suggest that, such as other adhesion molecules, T cell VLA integrins may also function in a dual capacity as adhesion and signalling molecules. In addition, they suggest that the interaction of T cells in vivo with ECM via VLA integrins plays a role not only in T cell migratory processes but may also influence Ag-specific T cell recognition.     

Rotational dynamics of the Fc epsilon receptor on mast cells monitored by specific monoclonal antibodies and IgE.

The rotational motions of the type I receptor for the Fc epsilon domains (Fc epsilon RI) present on mast cells were investigated by measuring the phosphorescence emission and anisotropy decay kinetics of erythrosin (Er) covalently bound to several Fc epsilon RI-specific macromolecular ligands. The latter consisted of three murine monoclonal antibodies (IgG class) raised against the Fc epsilon RI of rat mast cells (RBL-2H3 line), their Fab fragments, and a murine monoclonal IgE. Different anisotropy decay patterns were observed for the three monovalent Er-Fab fragments bound to the Fc epsilon RI, reflecting the rotational motion of the Fe epsilon RI reported by each specific macromolecular probe bound to its particular epitope. Internal motions of the tethered Er-labeled ligands may also contribute to the observed anisotropy decay, particularly in the case of cell-bound IgE. The results corroborate an earlier study with rat Er-IgE in which the Fc epsilon RI-IgE complex was shown to be mobile throughout the temperature range examined (5-37 degrees C). The anisotropy decays of the three Er-labeled, Fc epsilon RI-specific intact mAbs bound to cells also differed markedly. Whereas the decay curves of one mAb (H10) were characterized by temperature-dependent positive amplitudes and rather short rotational correlation times, the decay of a second mAb (J17) showed complex qualitative variations with temperature, and in the case of the third antibody (F4), there was no apparent decay of anisotropy over the time and temperature ranges examined.(ABSTRACT TRUNCATED AT 250 WORDS)