Expanding the Anti-Phl p 7 Antibody Toolkit: An Anti-Idiotype Nanobody Inhibitor.

We have previously produced a toolkit of antibodies, comprising recombinant human antibodies of all but one of the human isotypes, directed against the polcalcin family antigen Phl p 7. In this work, we complete the toolkit of human antibody isotypes with the IgD version of the anti-Phl p 7 monoclonal antibody. We also raised a set of nanobodies against the IgD anti-Phl p 7 antibody and identify and characterize one paratope-specific nanobody. This nanobody also binds to the IgE isotype of this antibody, which shares the same idiotype, and orthosterically inhibits the interaction with Phl p 7. The 2.1 Å resolution X-ray crystal structure of the nanobody in complex with the IgD Fab is described.

Crystal structures of the human IgD Fab reveal insights into CH1 domain diversity.

Antibodies of the IgD isotype remain the least well characterized of the mammalian immunoglobulin isotypes. Here we report three-dimensional structures for the Fab region of IgD, based on four different crystal structures, at resolutions of 1.45-2.75 Å. These IgD Fab crystals provide the first high-resolution views of the unique Cδ1 domain. Structural comparisons identify regions of conformational diversity within the Cδ1 domain, as well as among the homologous domains of Cα1, Cγ1 and Cµ1. The IgD Fab structure also possesses a unique conformation of the upper hinge region, which may contribute to the overall disposition of the very long linker sequence between the Fab and Fc regions found in human IgD. Structural similarities observed between IgD and IgG, and differences with IgA and IgM, are consistent with predicted evolutionary relationships for the mammalian antibody isotypes.

Generation and Characterization of Native and Sialic Acid-Deficient IgE.

Efficient characterization of IgE antibodies and their glycan structures is required for understanding their function in allergy and in the emerging AllergoOncology field for antibody immunotherapy. We report the generation, glyco-profiling and functional analysis of native and sialic acid-deficient glyco-engineered human IgE. The antibodies produced from human embryonic kidney cells were purified via a human IgE class-specific affinity matrix and structural integrity was confirmed by SDS-PAGE and size-exclusion chromatography (SEC). Purified IgEs specific for the tumor-associated antigens Chondroitin Sulfate Proteoglycan 4 (CSPG4-IgE) and Human Epidermal Growth Factor Receptor 2 (HER2-IgE) were devoid of by-products such as free light chains. Using neuraminidase-A, we generated sialic acid-deficient CSPG4-IgE as example glyco-engineered antibody. Comparative glycan analyses of native and glyco-engineered IgEs by Hydrophilic interaction liquid chromatography (HILIC)-high performance liquid chromatography (HPLC) indicated loss of sialic acid terminal residues and differential glycan profiles. Native and glyco-engineered CSPG4-IgEs recognized Fc receptors on the surface of human FcεRI-expressing rat basophilic leukemia RBL-SX38 cells, and of CD23/FcεRII-expressing human RPMI-8866 B-lymphocytes and bound to CSPG4-expressing A2058 human melanoma cells, confirming Fab-mediated recognition. When cross-linked on the cell surface, both IgEs triggered RBL-SX38 degranulation. We demonstrate efficient generation and functional competence of recombinant native and sialic acid-deficient IgEs.

Reviving lost binding sites: Exploring calcium-binding site transitions between human and murine CD23.

Immunoglobulin E (IgE) is a central regulatory and triggering molecule of allergic immune responses. IgE's interaction with CD23 modulates both IgE production and functional activities.CD23 is a noncanonical immunoglobulin receptor, unrelated to receptors of other antibody isotypes. Human CD23 is a calcium-dependent (C-type) lectin-like domain that has apparently lost its carbohydrate-binding capability. The calcium-binding site classically required for carbohydrate binding in C-type lectins is absent in human CD23 but is present in the murine molecule. To determine whether the absence of this calcium-binding site affects the structure and function of human CD23, CD23 mutant proteins with increasingly "murine-like" sequences were generated. Restoration of the calcium-binding site was confirmed by NMR spectroscopy, and structures of mutant human CD23 proteins were determined by X-ray crystallography, although no electron density for calcium was observed. This study offers insights into the evolutionary differences between murine and human CD23 and some of the functional differences between CD23 in different species.

Engineering the Fab fragment of the anti-IgE omalizumab to prevent Fab crystallization and permit IgE-Fc complex crystallization.

Immunoglobulin E (IgE) plays a central role in the allergic response, in which cross-linking of allergen by FcεRI-bound IgE triggers mast cell and basophil degranulation and the release of inflammatory mediators. The high-affinity interaction between IgE and FcεRI is a long-standing target for therapeutic intervention in allergic disease. Omalizumab is a clinically approved anti-IgE monoclonal antibody that binds to free IgE, also with high affinity, preventing its interaction with FcεRI. All attempts to crystallize the pre-formed complex between the omalizumab Fab and the Fc region of IgE (IgE-Fc), to understand the structural basis for its mechanism of action, surprisingly failed. Instead, the Fab alone selectively crystallized in different crystal forms, but their structures revealed intermolecular Fab/Fab interactions that were clearly strong enough to disrupt the Fab/IgE-Fc complexes. Some of these interactions were common to other Fab crystal structures. Mutations were therefore designed to disrupt two recurring packing interactions observed in the omalizumab Fab crystal structures without interfering with the ability of the omalizumab Fab to recognize IgE-Fc; this led to the successful crystallization and subsequent structure determination of the Fab/IgE-Fc complex. The mutagenesis strategy adopted to achieve this result is applicable to other intractable Fab/antigen complexes or systems in which Fabs are used as crystallization chaperones.

IgE Antibodies: From Structure to Function and Clinical Translation.

Immunoglobulin E (IgE) antibodies are well known for their role in mediating allergic reactions, and their powerful effector functions activated through binding to Fc receptors FcεRI and FcεRII/CD23. Structural studies of IgE-Fc alone, and when bound to these receptors, surprisingly revealed not only an acutely bent Fc conformation, but also subtle allosteric communication between the two distant receptor-binding sites. The ability of IgE-Fc to undergo more extreme conformational changes emerged from structures of complexes with anti-IgE antibodies, including omalizumab, in clinical use for allergic disease; flexibility is clearly critical for IgE function, but may also be exploited by allosteric interference to inhibit IgE activity for therapeutic benefit. In contrast, the power of IgE may be harnessed to target cancer. Efforts to improve the effector functions of therapeutic antibodies for cancer have almost exclusively focussed on IgG1 and IgG4 subclasses, but IgE offers an extremely high affinity for FcεRI receptors on immune effector cells known to infiltrate solid tumours. Furthermore, while tumour-resident inhibitory Fc receptors can modulate the effector functions of IgG antibodies, no inhibitory IgE Fc receptors are known to exist. The development of tumour antigen-specific IgE antibodies may therefore provide an improved immune functional profile and enhanced anti-cancer efficacy. We describe proof-of-concept studies of IgE immunotherapies against solid tumours, including a range of in vitro and in vivo evaluations of efficacy and mechanisms of action, as well as ex vivo and in vivo safety studies. The first anti-cancer IgE antibody, MOv18, the clinical translation of which we discuss herein, has now reached clinical testing, offering great potential to direct this novel therapeutic modality against many other tumour-specific antigens. This review highlights how our understanding of IgE structure and function underpins these exciting clinical developments.

Structure of a patient-derived antibody in complex with allergen reveals simultaneous conventional and superantigen-like recognition.

Antibodies classically bind antigens via their complementarity-determining regions, but an alternative mode of interaction involving V-domain framework regions has been observed for some B cell "superantigens." We report the crystal structure of an antibody employing both modes of interaction simultaneously and binding two antigen molecules. This human antibody from an allergic individual binds to the grass pollen allergen Phl p 7. Not only are two allergen molecules bound to each antibody fragment (Fab) but also each allergen molecule is bound by two Fabs: One epitope is recognized classically, the other in a superantigen-like manner. A single allergen molecule thus cross-links two identical Fabs, contrary to the one-antibody-one-epitope dogma, which dictates that a dimeric allergen at least is required for this to occur. Allergens trigger immediate hypersensitivity reactions by cross-linking receptor-bound IgE molecules on effector cells. We found that monomeric Phl p 7 induced degranulation of basophils sensitized solely with this monoclonal antibody expressed as an IgE, demonstrating that the dual specificity has functional consequences. The monomeric state of Phl p 7 and two structurally related allergens was confirmed by size-exclusion chromatography and multiangle laser light scattering, and the results were supported by degranulation studies with the related allergens, a second patient-derived allergen-specific antibody lacking the nonclassical binding site, and mutagenesis of the nonclassically recognized allergen epitope. The antibody dual reactivity and cross-linking mechanism not only have implications for understanding allergenicity and allergen potency but, importantly, also have broader relevance to antigen recognition by membrane Ig and cross-linking of the B cell receptor.

Structural basis for selective inhibition of immunoglobulin E-receptor interactions by an anti-IgE antibody.

Immunoglobulin E (IgE) antibodies play a central role in the allergic response: interaction with FcεRI on mast cells and basophils leads to immediate hypersensitivity reactions upon allergen challenge, while interaction with CD23/FcεRII, expressed on a variety of cells, regulates IgE synthesis among other activities. The receptor-binding IgE-Fc region has recently been found to display remarkable flexibility, from acutely bent to extended conformations, with allosteric communication between the distant FcεRI and CD23 binding sites. We report the structure of an anti-IgE antibody Fab (8D6) bound to IgE-Fc through a mixed protein-carbohydrate epitope, revealing further flexibility and a novel extended conformation with potential relevance to that of membrane-bound IgE in the B cell receptor for antigen. Unlike the earlier, clinically approved anti-IgE antibody omalizumab, 8D6 inhibits binding to FcεRI but not CD23; the structure reveals how this discrimination is achieved through both orthosteric and allosteric mechanisms, supporting therapeutic strategies that retain the benefits of CD23 binding.

Crystal structures of murine and human Histamine-Releasing Factor (HRF/TCTP) and a model for HRF dimerisation in mast cell activation.

In allergic disease, mast cell activation is conventionally triggered by allergen-mediated cross-linking of receptor-bound IgE on the cell surface. In addition to its diverse range of intracellular roles in apoptosis, cell proliferation and cancer, Histamine-Releasing Factor (HRF) also activates mast cells and basophils. A subset of IgE antibodies bind HRF through their Fab regions, and two IgE binding sites on HRF have been mapped. HRF can form dimers, and a disulphide-linked dimer is critical for activity. The current model for the activity of HRF in mast cell activation involves cross-linking of receptor-bound IgE by dimeric HRF, mediated by HRF/Fab interactions. HRF crystal and solution structures have provided little insight into either the formation of disulphide-linked HRF dimers or the ability of HRF to activate mast cells. We report the first crystal structure of murine HRF (mHRF) to 4.0Å resolution, revealing a conserved fold. We also solved the structure of human HRF (hHRF) in two new crystal forms, one at the highest resolution (1.4Å) yet reported. The high resolution hHRF structure reveals a disulphide-linked dimer, in which the two molecules are closely associated, and provides a model for the role of both human and murine HRF in mast cell activation.

IgE Trimers Drive SPE-7 Cytokinergic Activity.

Degranulation of mast cells and basophils, with release of agents of the allergic response, ensues when multivalent antigens bind to and cross-link the cells' receptor-bound IgE antibodies. A widely used commercial monoclonal IgE antibody, SPE-7 IgE from Sigma, was found to possess the radically anomalous property, termed "cytokinergic", of inducing basophil degranulation without the intervention of an antigen. We show here that the IgE monomer, freed of protein contaminants, is devoid of this activity, and that the source of the anomaly is a trace impurity, identified as a dissociation-resistant IgE trimer. Possible models for the formation of IgE trimers and the manner in which they cross-link cell surface receptors are suggested herein.

Thermal sensitivity and flexibility of the Cε3 domains in immunoglobulin E.

Immunoglobulin E (IgE) is the antibody that plays a central role in the mechanisms of allergic diseases such as asthma. Interactions with its receptors, FcεRI on mast cells and CD23 on B cells, are mediated by the Fc region, a dimer of the Cε2, Cε3 and Cε4 domains. A sub-fragment lacking the Cε2 domains, Fcε3-4, also binds to both receptors, although receptor binding almost exclusively involves the Cε3 domains. This domain also contains the N-linked glycosylation site conserved in other isotypes. We report here the crystal structures of IgE-Fc and Fcε3-4 at the highest resolutions yet determined, 1.75Šand 2.0Šrespectively, revealing unprecedented detail regarding the carbohydrate and its interactions with protein domains. Analysis of the crystallographic B-factors of these, together with all earlier IgE-Fc and Fcε3-4 structures, shows that the Cε3 domains exhibit the greatest intrinsic flexibility and quaternary structural variation within IgE-Fc. Intriguingly, both well-ordered carbohydrate and disordered polypeptide can be seen within the same Cε3 domain. A simplified method for comparing the quaternary structures of the Cε3 domains in free and receptor-bound IgE-Fc structures is presented, which clearly delineates the FcεRI and CD23 bound states. Importantly, differential scanning fluorimetric analysis of IgE-Fc and Fcε3-4 identifies Cε3 as the domain most susceptible to thermally-induced unfolding, and responsible for the characteristically low melting temperature of IgE.

Allosteric mechanism of action of the therapeutic anti-IgE antibody omalizumab.

Immunoglobulin E and its interactions with receptors FcϵRI and CD23 play a central role in allergic disease. Omalizumab, a clinically approved therapeutic antibody, inhibits the interaction between IgE and FcϵRI, preventing mast cell and basophil activation, and blocks IgE binding to CD23 on B cells and antigen-presenting cells. We solved the crystal structure of the complex between an omalizumab-derived Fab and IgE-Fc, with one Fab bound to each Cϵ3 domain. Free IgE-Fc adopts an acutely bent structure, but in the complex it is only partially bent, with large-scale conformational changes in the Cϵ3 domains that inhibit the interaction with FcϵRI. CD23 binding is inhibited sterically due to overlapping binding sites on each Cϵ3 domain. Studies of omalizumab Fab binding in solution demonstrate the allosteric basis for FcϵRI inhibition and, together with the structure, reveal how omalizumab may accelerate dissociation of receptor-bound IgE from FcϵRI, exploiting the intrinsic flexibility and allosteric potential of IgE.

Room temperature structure of human IgG4-Fc from crystals analysed in situ.

The Fc region of IgG antibodies (Cγ2 and Cγ3 domains) is responsible for effector functions such as antibody-dependent cell-mediated cytotoxicity and phagocytosis, through engagement with Fcγ receptors, although the ability to elicit these functions differs between the four human IgG subclasses. A key determinant of Fcγ receptor interactions is the FG loop in the Cγ2 domain. High resolution cryogenic IgG4-Fc crystal structures have revealed a unique conformation for this loop, which could contribute to the particular biological properties of this subclass. To further explore the conformation of the IgG4 Cγ2 FG loop at near-physiological temperature, we solved a 2.7Šresolution room temperature structure of recombinant human IgG4-Fc from crystals analysed in situ. The Cγ2 FG loop in one chain differs from the cryogenic structure, and adopts the conserved conformation found in IgG1-Fc; however, this conformation participates in extensive crystal packing interactions. On the other hand, at room temperature, and free from any crystal packing interactions, the Cγ2 FG loop in the other chain adopts the conformation previously observed in the cryogenic IgG4-Fc structures, despite both conformations being accessible. The room temperature human IgG4-Fc structure thus provides a more complete and physiologically relevant description of the conformation of this functionally critical Cγ2 FG loop.

Antibodies and superantibodies in patients with chronic rhinosinusitis with nasal polyps.

BACKGROUND: Chronic rhinosinusitis with nasal polyps is associated with local immunoglobulin hyperproduction and the presence of IgE antibodies against Staphylococcus aureus enterotoxins (SAEs). Aspirin-exacerbated respiratory disease is a severe form of chronic rhinosinusitis with nasal polyps in which nearly all patients express anti-SAEs. OBJECTIVES: We aimed to understand antibodies reactive to SAEs and determine whether they recognize SAEs through their complementarity-determining regions (CDRs) or framework regions. METHODS: Labeled staphylococcal enterotoxin (SE) A, SED, and SEE were used to isolate single SAE-specific B cells from the nasal polyps of 3 patients with aspirin-exacerbated respiratory disease by using fluorescence-activated cell sorting. Recombinant antibodies with "matched" heavy and light chains were cloned as IgG1, and those of high affinity for specific SAEs, assayed by means of ELISA and surface plasmon resonance, were recloned as IgE and antigen-binding fragments. IgE activities were tested in basophil degranulation assays. RESULTS: Thirty-seven SAE-specific, IgG- or IgA-expressing B cells were isolated and yielded 6 anti-SAE clones, 2 each for SEA, SED, and SEE. Competition binding assays revealed that the anti-SEE antibodies recognize nonoverlapping epitopes in SEE. Unexpectedly, each anti-SEE mediated SEE-induced basophil degranulation, and IgG1 or antigen-binding fragments of each anti-SEE enhanced degranulation by the other anti-SEE. CONCLUSIONS: SEEs can activate basophils by simultaneously binding as antigens in the conventional manner to CDRs and as superantigens to framework regions of anti-SEE IgE in anti-SEE IgE-FcεRI complexes. Anti-SEE IgG1s can enhance the activity of anti-SEE IgEs as conventional antibodies through CDRs or simultaneously as conventional antibodies and as "superantibodies" through CDRs and framework regions to SEEs in SEE-anti-SEE IgE-FcεRI complexes.

IgG4 Characteristics and Functions in Cancer Immunity.

IgG4 is the least abundant subclass of IgG in normal human serum, but elevated IgG4 levels are triggered in response to a chronic antigenic stimulus and inflammation. Since the immune system is exposed to tumor-associated antigens over a relatively long period of time, and tumors notoriously promote inflammation, it is unsurprising that IgG4 has been implicated in certain tumor types. Despite differing from other IgG subclasses by only a few amino acids, IgG4 possesses unique structural characteristics that may be responsible for its poor effector function potency and immunomodulatory properties. We describe the unique attributes of IgG4 that may be responsible for these regulatory functions, particularly in the cancer context. We discuss the inflammatory conditions in tumors that support IgG4, the emerging and proposed mechanisms by which IgG4 may contribute to tumor-associated escape from immune surveillance and implications for cancer immunotherapy.

Human IgG4: a structural perspective.

IgG4, the least represented human IgG subclass in serum, is an intriguing antibody with unique biological properties, such as the ability to undergo Fab-arm exchange and limit immune complex formation. The lack of effector functions, such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, is desirable for therapeutic purposes. IgG4 plays a protective role in allergy by acting as a blocking antibody, and inhibiting mast cell degranulation, but a deleterious role in malignant melanoma, by impeding IgG1-mediated anti-tumor immunity. These findings highlight the importance of understanding the interaction between IgG4 and Fcγ receptors. Despite a wealth of structural information for the IgG1 subclass, including complexes with Fcγ receptors, and structures for intact antibodies, high-resolution crystal structures were not reported for IgG4-Fc until recently. Here, we highlight some of the biological properties of human IgG4, and review the recent crystal structures of IgG4-Fc. We discuss the unexpected conformations adopted by functionally important Cγ2 domain loops, and speculate about potential implications for the interaction between IgG4 and FcγRs.

Structure and dynamics of IgE-receptor interactions: FcεRI and CD23/FcεRII.

Immunoglobulin E (IgE) is well known for its role in allergic disease, the manifestations of which are mediated through its two Fc receptors, FcεRI and CD23 (FcεRII). IgE and its interactions with these receptors are therefore potential targets for therapeutic intervention, and exciting progress has been made in this direction. Furthermore, recent structural studies of IgE-Fc, the two receptors, and of their complexes, have revealed a remarkable degree of plasticity at the IgE-CD23 interface and an even more remarkable degree of dynamic flexibility within the IgE molecule. Indeed, there is allosteric communication between the two receptor-binding sites, which we now know are located at some distance from each other in IgE-Fc (at opposite ends of the Cε3 domain). The conformational changes associated with FcεRI and CD23 binding to IgE-Fc ensure that their interactions are mutually incompatible, and it may be that this functional imperative has driven IgE to evolve such a dynamic structure. Appreciation of these new structural data has revised our view of IgE structure, shed light on the co-evolution of antibodies and their receptors, and may open up new therapeutic opportunities.

Crystal structure of deglycosylated human IgG4-Fc.

The Fc region of IgG antibodies, important for effector functions such as antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis and complement activation, contains an oligosaccharide moiety covalently attached to each C(H)2 domain. The oligosaccharide not only orients the C(H)2 domains but plays an important role in influencing IgG effector function, and engineering the IgG-Fc oligosaccharide moiety is an important aspect in the design of therapeutic monoclonal IgG antibodies. Recently we reported the crystal structure of glycosylated IgG4-Fc, revealing structural features that could explain the anti-inflammatory biological properties of IgG4 compared with IgG1. We now report the crystal structure of enzymatically deglycosylated IgG4-Fc, derived from human serum, at 2.7Šresolution. Intermolecular C(H)2-C(H)2 domain interactions partially bury the C(H)2 domain surface that would otherwise be exposed by the absence of oligosaccharide, and two Fc molecules are interlocked in a symmetric, open conformation. The conformation of the C(H)2 domain DE loop, to which oligosaccharide is attached, is altered in the absence of carbohydrate. Furthermore, the C(H)2 domain FG loop, important for Fcγ receptor and C1q binding, adopts two different conformations. One loop conformation is unique to IgG4 and would disrupt binding, consistent with IgG4's anti-inflammatory properties. The second is similar to the conserved conformation found in IgG1, suggesting that in contrast to IgG1, the IgG4 C(H)2 FG loop is dynamic. Finally, crystal packing reveals a hexameric arrangement of IgG4-Fc molecules, providing further clues about the interaction between C1q and IgG.

Dynamics of inter-heavy chain interactions in human immunoglobulin G (IgG) subclasses studied by kinetic Fab arm exchange.

Interdomain interactions between the CH3 domains of antibody heavy chains are the first step in antibody assembly and are of prime importance for maintaining the native structure of IgG. For human IgG4 it was shown that CH3-CH3 interactions are weak, resulting in the potential for half-molecule exchange ("Fab arm exchange"). Here we systematically investigated non-covalent interchain interactions for CH3 domains in the other human subclasses, including polymorphisms (allotypes), using real-time monitoring of Fab arm exchange with a FRET-based kinetic assay. We identified structural variation between human IgG subclasses and allotypes at three amino acid positions (Lys/Asn-392, Val/Met-397, Lys/Arg-409) to alter the strength of inter-domain interactions by >6 orders of magnitude. Each substitution affected the interactions independent from the other substitutions in terms of affinity, but the enthalpic and entropic contributions were non-additive, suggesting a complex interplay. Allotypic variation in IgG3 resulted in widely different CH3 interaction strengths that were even weaker for IgG3 than for IgG4 in the case of allotype G3m(c3c5*/6,24*), whereas G3m(s*/15*) was equally stable to IgG1. These interactions are sufficiently strong to maintain the structural integrity of IgG1 during its normal life span; for IgG2 and IgG3 the inter-heavy chain disulfide bonds are essential to prevent half-molecule dissociation, whereas the labile hinge disulfide bonds favor half-molecule exchange in vivo for IgG4.

Structural determinants of unique properties of human IgG4-Fc.

Human IgG4, normally the least abundant of the four subclasses of IgG in serum, displays a number of unique biological properties. It can undergo heavy-chain exchange, also known as Fab-arm exchange, leading to the formation of monovalent but bispecific antibodies, and it interacts poorly with FcγRII and FcγRIII, and complement. These properties render IgG4 relatively "non-inflammatory" and have made it a suitable format for therapeutic monoclonal antibody production. However, IgG4 is also known to undergo Fc-mediated aggregation and has been implicated in auto-immune disease pathology. We report here the high-resolution crystal structures, at 1.9 and 2.35 Å, respectively, of human recombinant and serum-derived IgG4-Fc. These structures reveal conformational variability at the CH3-CH3 interface that may promote Fab-arm exchange, and a unique conformation for the FG loop in the CH2 domain that would explain the poor FcγRII, FcγRIII and C1q binding properties of IgG4 compared with IgG1 and -3. In contrast to other IgG subclasses, this unique conformation folds the FG loop away from the CH2 domain, precluding any interaction with the lower hinge region, which may further facilitate Fab-arm exchange by destabilisation of the hinge. The crystals of IgG4-Fc also display Fc-Fc packing contacts with very extensive interaction surfaces, involving both a consensus binding site in IgG-Fc at the CH2-CH3 interface and known hydrophobic aggregation motifs. These Fc-Fc interactions are compatible with intact IgG4 molecules and may provide a model for the formation of aggregates of IgG4 that can cause disease pathology in the absence of antigen.