Jenny M. Woof

The function of immunoglobulin A in immunity.

The vast surfaces of the gastrointestinal, respiratory, and genitourinary tracts represent major sites of potential attack by invading micro‐organisms. Immunoglobulin A (IgA), as the principal antibody class in the secretions that bathe these mucosal surfaces, acts as an important first line of defence. IgA, also an important serum immunoglobulin, mediates a variety of protective functions through interaction with specific receptors and immune mediators. The importance of such protection is underlined by the fact that certain pathogens have evolved mechanisms to compromise IgA‐mediated defence, providing an opportunity for more effective invasion. IgA function may also be perturbed in certain disease states, some of which are characterized by deposition of IgA in specific tissues. This review details current understanding of the roles played by IgA in both health and disease. Copyright

Human antibody–Fc receptor interactions illuminated by crystal structures

Immunoglobulins couple the recognition of invading pathogens with the triggering of potent effector mechanisms for pathogen elimination. Different immunoglobulin classes trigger different effector mechanisms through interaction of immunoglobulin Fc regions with specific Fc receptors (FcRs) on immune cells. Here, we review the structural information that is emerging on three human immunoglobulin classes and their FcRs. New insights are provided, including an understanding of the antibody conformational adjustments that are required to bring effector cell and target cell membranes sufficiently close for efficient killing and signal transduction to occur. The results might also open up new possibilities for the design of therapeutic antibodies.

Human Antibody Effector Function

Publisher Summary This chapter focuses on human antibody effector function. A molecular explanation of antibody effector function requires the description of multiple antibody molecules cross-linking an array of antigen molecules to multiple effector molecules. The antigen molecules are likely to be on a cell surface and the effector molecules are either large, as for complement, or on a cell surface, as for fragment crystallizable (Fc) receptors. The chapter presents crystal structures for (1) Fab fragments, (2) Fab fragments complexed with a number of antigens, and (3) Fc from immunoglobulin (IgG). It also has the low-resolution structures of two mutant whole IgG molecules. These mutant molecules lack the hinge region and generally show poor effector activity. They crystallize and show a complete diffraction pattern because the loss of the hinge reduces their flexibility. The crystal structures of human and rabbit Fc from IgG are determined to intermediate resolution and analyzed in detail in terms of potential interacting sites. The chapter discusses complement activation by antibodies. The complement cascade can be usefully divided into two phases: (1) the deposition of C3 on the antigenic cell surface, thereby facilitating phagocytosis, in the classical pathway this requires the sequential activation of C1 and C42, (2) the formation of the membrane attack complex resulting in cell lysis, this requires the activation of C5 and attachment of C6–9.

Identification of Residues in the CH2/CH3 Domain Interface of IgA Essential for Interaction with the Human Fcα Receptor (FcαR) CD89

Cellular receptors for IgA (FcαR) mediate important protective functions. An extensive panel of site-directed mutant IgAs was used to identify IgA residues critical for FcαR (CD89) binding and triggering. Although a tailpiece-deleted IgA1 was able to bind and trigger CD89, antibodies featuring CH3 domain exchanges between human IgA1 and IgG1 could not, indicating that both domains but not the tailpiece are required for FcαR recognition. To further investigate the role of the interdomain region, numerous IgA1s, each with a point substitution in either of two interdomain loops (Leu-257—Gly-259 in Cα2; Pro-440—Phe-443 in Cα3), were generated. With only one exception (G259R), substitutions produced either ablation (L257R, P440A, A442R, F443R) or marked reduction (P440R) in CD89 binding and triggering. Further support for involvement of these interdomain loops was provided by interspecies comparisons of IgA. Thus a human IgA1 mutant, LA441–442MN, which mimicked the mouse IgA loop sequence through substitution of two adjacent residues in the Cα3 loop, was found, like mouse IgA, not to bind CD89. In contrast, bovine IgA1, identical to human IgA1 within these interdomain loops despite numerous differences elsewhere in the Fc region, did bind CD89. We have thus identified motifs in the interdomain region of IgA Fc critical for FcαR binding and triggering, significantly enhancing present understanding of the molecular basis of the IgA-FcαR interaction.

Structure and function relationships in IgA

Immunoglobulin A (IgA) has a critical role in immune defense particularly at the mucosal surfaces, and is equipped to do so by the unique structural attributes of its heavy chain and by its ability to polymerize. Here, we provide an overview of human IgA structure, describing the distinguishing features of the IgA1 and IgA2 subclasses and mapping the sites of interaction with host receptors important for IgAs functional repertoire. Remarkably, these same interaction sites are targeted by binding proteins and proteases produced by various pathogens as a means to subvert the protective IgA response. As interest in the prospect of therapeutic IgA-based monoclonal antibodies grows, the emerging understanding of the relationship between IgA structure and function will be invaluable for maximizing the potential of these novel reagents.

Streptococcal IgA-binding Proteins Bind in the Cα2-Cα3 Interdomain Region and Inhibit Binding of IgA to Human CD89

Certain pathogenic bacteria express surface proteins that bind to the Fc part of human IgA or IgG. These bacterial proteins are important as immunochemical tools and model systems, but their biological function is still unclear. Here, we describe studies of three streptococcal proteins that bind IgA: the Sir22 and Arp4 proteins of Streptococcus pyogenes and the unrelated β protein of group B streptococcus. Analysis of IgA domain swap and point mutants indicated that two loops at the Cα2/Cα3 domain interface are critical for binding of the streptococcal proteins. This region is also used in binding the human IgA receptor CD89, an important mediator of IgA effector function. In agreement with this finding, the three IgA-binding proteins and a 50-residue IgA-binding peptide derived from Sir22 blocked the ability of IgA to bind CD89. Further, the Arp4 protein inhibited the ability of IgA to trigger a neutrophil respiratory burst via CD89. Thus, we have identified residues on IgA-Fc that play a key role in binding of different streptococcal IgA-binding proteins, and we have identified a mechanism by which a bacterial IgA-binding protein may interfere with IgA effector function.

A Novel Human IgA Monoclonal Antibody Protects against Tuberculosis

Abs have been shown to be protective in passive immunotherapy of tuberculous infection using mouse experimental models. In this study, we report on the properties of a novel human IgA1, constructed using a single-chain variable fragment clone (2E9), selected from an Ab phage library. The purified Ab monomer revealed high binding affinities for the mycobacterial α-crystallin Ag and for the human FcαRI (CD89) IgA receptor. Intranasal inoculations with 2E9IgA1 and recombinant mouse IFN-γ significantly inhibited pulmonary H37Rv infection in mice transgenic for human CD89 but not in CD89-negative littermate controls, suggesting that binding to CD89 was necessary for the IgA-imparted passive protection. 2E9IgA1 added to human whole-blood or monocyte cultures inhibited luciferase-tagged H37Rv infection although not for all tested blood donors. Inhibition by 2E9IgA1 was synergistic with human rIFN-γ in cultures of purified human monocytes but not in whole-blood cultures. The demonstration of the mandatory role of FcαRI (CD89) for human IgA-mediated protection is important for understanding of the mechanisms involved and also for translation of this approach toward development of passive immunotherapy of tuberculosis.

Isolation and Detection of Human IgA Using a Streptococcal IgA-Binding Peptide

Bacterial proteins that bind to the Fc part of IgG have found widespread use in immunology. A similar protein suitable for the isolation and detection of human IgA has not been described. Here, we show that a 50-residue synthetic peptide, designated streptococcal IgA-binding peptide (Sap) and derived from a streptococcal M protein, can be used for single-step affinity purification of human IgA. High affinity binding of IgA required the presence in Sap of a C-terminal cysteine residue, not present in the intact M protein. Passage of human serum through a Sap column caused depletion of >99% of the IgA, and elution of the column allowed quantitative recovery of highly purified IgA, for which the proportions of the IgA1 and IgA2 subclasses were the same as in whole serum. Moreover, immobilized Sap could be used for single-step purification of secretory IgA of both subclasses from human saliva, with a recovery of ∼45%. The Sap peptide could also be used to specifically detect IgA bound to Ag. Together, these data indicate that Sap is a versatile Fc-binding reagent that may open new possibilities for the characterization of human IgA.

Fc receptors and immunity to parasites

Fc receptors (FcRs) are crucial in the immune system; they mediate a plethora of biological functions as diverse as antigen presentation, phagocytosis, cytotoxicity, induction of inflammatory cascades and modulation of immune responses. Parasites, in order to survive in the immunocompetent host, have devised ingenious methods to subvert this important aspect of the immune response. This article discusses the current thinking on FcRs, their role in immunity to parasites, and immune evasion strategies employed by parasites in their attempt to neutralize the important immune defense mechanisms mediated by these molecules.

IgA function – variations on a theme

JENNY M. WOOF* & MICHAEL A. KERR *Division of Pathology and Neuroscience, University of Dundee MedicalSchool, Ninewells Hospital, Dundee and Department of Clinical Biochemistry and Immunology, General Infirmary at Leeds,Leeds, UKImmunoglobulin A (IgA), as the major class of antibodypresent in the mucosal secretions of most mammals, rep-resents a key first line of defence against invasion by inhaledand ingested pathogens at the vulnerable mucosal surfaces.IgA is also found at significant concentrations in the serumof many species, where it functions as a second line ofdefence mediating elimination of pathogens that havebreached the mucosal surface. Receptors specific for the Fcregion of IgA, FcaR, are key mediators of IgA effectorfunction and studies described in this issue of Immunologyby Rogers and colleagues