Identification of residues within the extracellular domain 1 of bovine Fc gamma 2R essential for binding bovine IgG2

Neutrophils and monocytes in cattle express a novel class of immunoglobulin Fc receptor, specific for bovine IgG2 (bIgG2), termed bFc gamma 2R. In cows, the ability of neutrophils to kill immunoglobulin-opsonized microorganisms appears to depend largely on this subclass, whose interaction with bFc gamma 2R initiates the killing process. bFc gamma 2R is a transmembrane glycoprotein consisting of two extracellular immunoglobulin-like domains, followed by a 19-amino acid membrane-spanning region and a short cytoplasmic tail. Although related to other mammalian Fc gamma Rs, bFc gamma 2R belongs to a novel gene family that includes the human killer cell inhibitory receptor and Fc alpha RI (CD89) proteins. We have shown previously (Morton, H. C., van Zandbergen, G., van Kooten, C., Howard, C. J., van de Winkel, J. G., and Brandtzaeg, P. (1999) J. Exp. Med. 189, 1715-1722) that like these proteins (and unlike other Fc gamma Rs), bFc gamma 2R binds bIgG2 via the membrane-distal extracellular domain 1 (EC1). In this present study, we introduced mutations into the predicted loop regions of the EC1 domain and assayed the resulting bFc gamma 2R mutants for their ability to bind bIgG2. Our results indicated that the bIgG2 binding site lies within the predicted F-G loop region of the EC1 domain. Furthermore, single amino acid mutational analysis of this region identified Phe-82 and Trp-87 as being critical for bIgG2 binding.     

Identification of residues in the CH2/CH3 domain interface of IgA essential for interaction with the human fcalpha receptor (FcalphaR) CD89

Cellular receptors for IgA (FcalphaR) mediate important protective functions. An extensive panel of site-directed mutant IgAs was used to identify IgA residues critical for FcalphaR (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 FcalphaR 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 Calpha2; Pro-440-Phe-443 in Calpha3), 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 Calpha3 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 FcalphaR binding and triggering, significantly enhancing present understanding of the molecular basis of the IgA-FcalphaR interaction.     

The human IgA-Fc alpha receptor interaction and its blockade by streptococcal IgA-binding proteins

IgA plays a key role in immune defence of the mucosal surfaces. IgA can trigger elimination mechanisms against pathogens through the interaction of its Fc region with Fc alpha Rs (receptors specific for the Fc region of IgA) present on neutrophils, macrophages, monocytes and eosinophils. The human Fc alpha R (CD89) shares homology with receptors specific for the Fc region of IgG (Fc gamma Rs) and IgE (Fc epsilon RIs), but is a more distantly related member of the receptor family. CD89 interacts with residues lying at the interface of the two domains of IgA Fc, a site quite distinct from the homologous regions at the top of IgG and IgE Fc recognized by Fc gamma R and Fc epsilon RI respectively. Certain pathogenic bacteria express surface proteins that bind to human IgA Fc. Experiments with domain-swap antibodies and mutant IgAs indicate that binding of three such proteins (Sir22 and Arp4 of Streptococcus pyogenes and beta protein of group B streptococci) depend on sites in the Fc interdomain region of IgA, the binding region also used by CD89. Further, we have found that the streptococcal proteins can inhibit interaction of IgA with CD89, and have thereby identified a mechanism by which a bacterial IgA-binding protein may modulate IgA effector function.     

A subset of human dendritic cells expresses IgA Fc receptor (CD89), which mediates internalization and activation upon cross-linking by IgA complexes

Immature dendritic cells (DC) sample Ags within nonlymphoid tissues and acquire exogenous proteins/pathogens via scavenger receptors or Ig FcR such as Fc gamma R and Fc epsilon R. IgA is present in a significant proportion among serum Ig and is the main isotype in mucosae, where DC are numerous. We found that a functional Fc alpha R (CD89) was expressed in situ and in vitro on interstitial-type DC but not on Langerhans cell-type DC. Interstitial-type DC expressed CD89 as a 50- to 75-kDa glycoprotein with a 32-kDa protein core, which was down-regulated upon addition of TGF-beta 1. DC, Fc alpha R specifically, bound IgA1 and IgA2. Cross-linking of CD89 on DC triggered endocytosis in time-dependent manner. In addition, internalization of polymeric IgA complexes induced the production of IL-10 and DC activation, as reflected by up-regulation of CD86 costimulatory molecules, class II MHC expression, and increased allostimulatory activity. Therefore, interstitial-type DC may use Fc alpha R-mediated Ag sampling in the subepithelium to check tissue integrity while Langerhans cells inside epithelial layers may neglect IgA immune complexes.     

Mouse IgA Fc receptor on CD3+ T cells. Molecular forms of IgA that bind to a 38-kDa Fc alpha R protein and development of an anti-Fc alpha R antisera

Previous studies have shown that splenic T cells from mice that bear IgA myelomas, as well as certain T cell lines, express receptors for the Fc of IgA, and are termed Fc alpha R. In this study, we have isolated and characterized two CD3+ T cell lines derived by fusion of murine Peyer's patch (PP) CD4+ T cells with the BW 5147 lymphoma cell line. These cell lines, designated PPT4-6 and PPT4-16, were shown to bind monomeric or dimeric IgA, whereas the fusion partner did not bind either form of IgA. However, polymeric IgA (m.w. 600,000) bound equally well to all three cell lines. Similar results were also obtained with two known Fc alpha R+ T cell lines, ThHA1 nos. 9 and 10. Immunoprecipitation studies with IgA on PPT4-16 and ThHA1 no. 9 have shown that IgA binds to a 38-kDa protein. A rabbit antiserum was prepared to a 38-kDa fraction of Fc alpha R+ T cell membranes, and heterophilic antibody was removed from the antiserum by adsorption with mouse thymocytes, BW 5147 and R1.1 lymphoma. The antiserum bound to both PPT4-16 and ThHA1 no. 9 as well as to other Fc alpha R+ T cells, but did not bind to thymocytes or to the T lymphomas R1.1 or BW 5147. The antiserum appeared specific for the Fc alpha R, because it failed to block binding of anti-CD3 (145 2C11) or other surface molecule-specific antibodies. Further, competitive inhibition studies with IgA and anti-Fc alpha R (38 kDa) showed that preincubation of Fc alpha R+ T cells with the anti-38-kDa protein completely eliminated IgA binding, whereas IgA partially blocked the binding of the anti-Fc alpha R antibodies to the cell membrane. Immunoisolation with the anti-Fc alpha R antibody of radioiodinated cell membrane proteins from Fc alpha R+ T cells, but not from Fc alpha R- cells, gave a distinct band at 38 kDa. To further test the specificity of this antiserum, we have isolated T cells from spleens of IgA-myeloma bearing mice, and tested the phenotype and IgA binding. A subset consisting of 15 to 20% of CD3+, CD8+ T cells was found that bound monomeric or dimeric IgA. Further, the anti-Fc alpha R antiserum also recognized this CD8+ T cell subset, and preincubation of the cells with antibody resulted in their failure to bind IgA. Our results indicate that the Fc alpha R on T cell lines derived from PP is a 38-kDa protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

CD229 (Ly9) lymphocyte cell surface receptor interacts homophilically through its N-terminal domain and relocalizes to the immunological synapse

CD229 is a member of the CD150 family of the Ig superfamily expressed on T and B cells. Receptors of this family regulate cytokine production and cytotoxicity of lymphocytes and NK cells. The cytoplasmic tail of CD229 binds to SAP, a protein that is defective in X-linked lymphoproliferative syndrome. To identify the CD229 ligand, we generated a soluble Ig fusion protein containing the two N-terminal extracellular domains of human CD229 (CD229-Ig). CD229-Ig bound to CD229-transfected cells, whereas no binding was detected on cells expressing other CD150 family receptors, showing that CD229 binds homophilically. Both human and mouse CD229 interacted with itself. Domain deletion mutants showed that the N-terminal Ig-domain mediates homophilic adhesion. CD229-CD229 binding was severely compromised when the charged amino acids E27 and E29 on the predicted B-C loop and R89 on the F-G loop of the N-terminal domain were mutated to alanine. In contrast, one mutation, R44A, enhanced the homophilic interaction. Confocal microscopy image analysis revealed relocalization of CD229 to the contact area of T and B cells during Ag-dependent immune synapse formation. Thus, CD229 is its own ligand and participates in the immunological synapse.     

C/EBP alpha and Ets protein family members regulate the human myeloid IgA Fc receptor (Fc alpha R,CD89) promoter

Fc alpha R (CD89), the FcR for IgA, is expressed exclusively in myeloid cells, including monocytes/macrophages, neutrophils, and eosinophils, and is thought to mediate IgA-triggered cellular functions in immunity. Here we demonstrate that the Fc alpha R 5'-flanking region from -102 to -64 relative to the ATG translation initiation codon is essential for promoter activity and contains two functional binding motifs for C/EBP and Ets family members at -74 and -92, respectively. EMSAs and cotransfection experiments show that C/EBP alpha acts as a major activator of the Fc alpha R promoter at least in immature myeloid cells. In addition, we found two additional functional targets of C/EBP alpha at -139 and -127. On the other hand, the Fc alpha R Ets binding motif could bind Elf-1 and mediate the trans-activation by cotransfected Elf-1, but a major component of the complex forming on this site appears to be an unidentified Ets-like nuclear protein that is preferentially detected in cells of hemopoietic origin. Furthermore, separation of the C/EBP and Ets binding sites reduces Fc alpha R promoter activity, suggesting some functional interaction between these factors. As the in vivo role of Fc alpha R is still incompletely defined, these findings reveal the features controlling the Fc alpha R promoter in myeloid lineage and provide a foundation for clarifying regulatory mechanisms of Fc alpha R gene expression associated with its potential roles.     

Pentraxins and IgA share a binding hot‐spot on FcαRI

The pentraxins, C‐reactive protein (CRP), and serum amyloid P component (SAP) have previously been shown to function as innate opsonins through interactions with Fcγ receptors. The molecular details of these interactions were elucidated by the crystal structure of SAP in complex with FcγRIIA. More recently, pentraxins were shown to bind and activate FcαRI (CD89), the receptor for IgA. Here, we used mutations of the receptor based on a docking model to further examine pentraxin recognition by FcαRI. The solution binding of pentraxins to six FcαRI alanine cluster mutants revealed that mutations Y35A and R82A, on the C‐and F‐strands of the D1 domain, respectively, markedly reduced receptor binding to CRP and SAP. These residues are in the IgA‐binding site of the receptor, and thus, significantly affected receptor binding to IgA. The shared pentraxin and IgA‐binding site on FcαRI is further supported by the results of a solution binding competition assay. In addition to the IgA‐binding site, pentraxins appear to interact with a broader region of the receptor as the mutation in the C′‐strand (R48A/E49A) enhanced pentraxin binding. Unlike Fcγ receptors, the H129A/I130A and R178A mutations on the BC‐ and FG‐loops of D2 domain, respectively, had little effect on FcαRI binding to the pentraxins. In conclusion, our data suggest that the pentraxins recognize a similar site on FcαRI as IgA.     

The interaction of Fc alpha RI with IgA and its implications for ligand binding by immunoreceptors of the leukocyte receptor cluster

This study defines the molecular basis of the FcalphaRI (CD89):IgA interaction, which is distinct from that of the other leukocyte Fc receptors and their Ig ligands. A comprehensive analysis using both cell-free (biosensor) and cell-based assays was used to define and characterize the IgA binding region of FcalphaRI. Biosensor analysis of mutant FcalphaRI proteins showed that residues Y35, Y81, and R82 were essential for IgA binding, and R52 also contributed. The role of the essential residues (Y35 and R82) was confirmed by analysis of mutant receptors expressed on the surface of mammalian cells. These receptors failed to bind IgA, but were detected by the mAb MY43, which blocks IgA binding to FcalphaRI, indicating that its epitope does not coincide with these IgA binding residues. A homology model of the ectodomains of FcalphaRI was generated based on the structures of killer Ig-like receptors, which share 30-34% identity with FcalphaRI. Key structural features of killer Ig-like receptors are appropriately reproduced in the model, including the structural conservation of the interdomain linker and hydrophobic core (residues V17, V97, and W183). In this FcalphaRI model the residues forming the IgA binding site identified by mutagenesis form a single face near the N-terminus of the receptor, distinct from other leukocyte Fc receptors where ligand binding is in the second domain. This taken together with major differences in kinetics and affinity for IgA:FcalphaRI interaction that were observed depending on whether FcalphaRI was immobilized or in solution suggest a mode of interaction unique among the leukocyte receptors.     

A competitive mechanism for staphylococcal toxin SSL7 inhibiting the leukocyte IgA receptor, Fc alphaRI, is revealed by SSL7 binding at the C alpha2/C alpha3 interface of IgA

Leukocyte recruitment and effector functions like phagocytosis and respiratory burst are key elements of immunity to infection. Pathogen survival is dependent upon the ability to overwhelm, evade or inhibit the immune system. Pathogenic group A and group B streptococci are well known to produce virulence factors that block the binding of IgA to the leukocyte IgA receptor, Fc alphaRI, thereby inhibiting IgA-mediated immunity. Recently we found Staphylococcus aureus also interferes with IgA-mediated effector functions as the putative virulence factor SSL7 also binds IgA and blocks binding to Fc alphaRI. Herein we report that SSL7 and Fc alphaRI bind many of the same key residues in the Fc region of human IgA. Residues Leu-257 and Leu-258 in domain C alpha2 and residues 440-443 PLAF in C alpha3 of IgA lie at the C alpha2/C alpha3 interface and make major contributions to the binding of both the leukocyte receptor Fc alphaRI and SSL7. It is remarkable this S. aureus IgA binding factor and unrelated factors from streptococci are functionally convergent, all targeting a number of the same residues in the IgA Fc, which comprise the binding site for the leukocyte IgA receptor, Fc alphaRI.     

Murine IgA binding factors produced by Fc alpha R(+) T cells: role of Fc gamma R(+) cells for the induction of Fc alpha R and formation of IgA-binding factor in Con A-activated cells

The relationship between the production of a T cell factor having affinity for IgA (IgA-binding factor(s); IgA BF) and the expression of Fc receptors specific for IgA (Fc alpha R) was studied by using murine spleen cells activated with concanavalin A (Con A blasts). Fc alpha R was detected by the cytophilic binding of anti-TNP murine IgA myeloma protein (MOPC 315 IgA) to Con A blasts as determined by an indirect rosette method with trinitrophenylated sheep red blood cells (TNP-SRBC). After 18 hr preculture with IgA, Fc alpha R was expressed on 15 to 20% of Con A blasts, which released IgA BF suppressing the in vitro IgA synthesis of the spleen cells stimulated with pokeweed mitogen (PWM). Without preculture with IgA, there was neither induction of Fc alpha R nor the production of IgA BF from Con A blasts. Fc alpha R was not induced on Con A blasts by IgA if Fc gamma R(+) cells were depleted from the blasts by rosetting with SRBC sensitized with rabbit IgG antibody (EA gamma). Even after preculture with IgA, the suppressive IgA BF was undetectable in the culture supernatant of Con A blasts depleted of the Fc gamma R(+) cell population. By using a double rosette method with EA gamma and trinitrophenylated quail red blood cells, Fc alpha R proved to be co-expressed on Fc gamma R(+) precursor T cells in the Con A blasts. The results suggested that both Fc gamma R and Fc alpha R could be co-expressed on Con A blasts, as is the case with T2D4 Fc gamma R(+), Fc alpha R(+) T hybridoma cells, which are known to produce IgG-binding factor(s) (IgG BF) and IgA BF. The relationship between Fc gamma R and Fc alpha R on a single cell was studied by using monoclonal anti-Fc gamma R antibody (2. 4G2 ). The reactivity of 2. 4G2 antibody with T cell Fc gamma R was proved by the inhibition of EA gamma rosette formation by Con A blasts or T2D4 cells. The addition of 2. 4G2 monoclonal antibody, however, did not affect the induction of Fc alpha R on Con A blasts by IgA. Furthermore, the binding of IgA to Fc alpha R already expressed on L5178Y T lymphoma cell line cells was not inhibited by the monoclonal antibody. The results confirmed that Fc alpha R are distinct from Fc gamma R co-expressed on the same Con A blasts, and that the expression of Fc alpha R on Fc gamma R(+) T cells and their production of suppressive IgA BF may be induced by the binding of IgA to Fc alpha R.     

Streptococcal IgA-binding proteins bind in the Calpha 2-Calpha 3 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 beta protein of group B streptococcus. Analysis of IgA domain swap and point mutants indicated that two loops at the Calpha2/Calpha3 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.     

Identification of the linear epitope for Fc-binding on the bovine IgG2 Fc receptor (boFcgamma2R) using synthetic peptides

To identify the linear epitope for Fc-binding on the bovine IgG2 Fc receptor (boFcgamma2R), peptides derived from the membrane-distal extracellular domain (EC1) of boFcgamma2R corresponding to the homologous region of human FcalphaRI were synthesized. Binding of bovine IgG2 to the different peptides was tested by Dot-blot assay, and the peptide showing maximal binding was further modified by truncation and mutation. The minimum effective peptide 82FIGV85 located in the putative F-G loop of the EC1 domain was found to bind bovine IgG2 specifically and inhibit the binding of bovine IgG2 to the receptor. The Phe82, Ile83 and Val85 residues within the linear epitope were shown to be critical for IgG2-binding. Such functional epitope peptide should be very useful for understanding the IgG-Fcgamma interaction and development of FcR-targeting drugs.     

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.     

Isotype-specific immunoregulation; characterization and function of Fc receptors on T-T hybridomas which produce murine IgA-binding factor

Several methods have been used in the present study to characterize Fc receptors (FcR) expressed on T-T hybridomas derived from mouse Peyer's patch T helper (Th) cell clones that preferentially support IgA responses. These T hybridomas (designated Th HA cells) produce IgA-binding factor (IBF alpha) which regulates antigen-dependent IgA responses. The ultrastructure of Th HA cells and the distribution of Fc alpha R on these cell lines were determined by colloidal gold (CG) immunoelectron microscopy (IEM). When Th HA cells were incubated with purified mouse IgA followed by CG-labeled anti-IgA, an even pattern of CG was distributed on the cell membrane. To ensure that binding occurred through Fc alpha R, Th HA cells were mixed with MOPC 315 IgA anti-DNP, followed by staining with CG-labeled TNP-human serum albumin. This resulted in an identical pattern of gold particle distribution, confirming expression of Fc alpha R on Th HA cells. No Fc mu R or Fc gamma 1R were detectable on Th HA cells by IEM. Immunocytoadherence with TNP-conjugated erythrocytes confirmed that Th HA cells were Fc alpha R+; however, no IgM or IgG rosettes were seen. When these cell lines were analyzed by flow cytometry (FACS) using IgA, IgM, or IgG1 and FITC-labeled anti-H chain-specific antibodies, 55 to 65% of cultured Th HA cells expressed Fc alpha R, and 11 to 18% expressed Fc mu R; however, no Fc gamma 1R was detectable on Th HA cells. The use of ELISA with Th HA cells as antigen confirmed the expression of Fc alpha R and the presence of less Fc mu R on these two cell lines. Solubilized membrane fractions derived from Th HA cells were tested for the presence of FcR by ELISA and for biologic function for support of IgA responses in Peyer's patch B cell cultures. Both Fc alpha R and Fc mu R were detected in fractions derived from Th HA cells. Furthermore, these fractions supported in vitro IgA anti-sheep erythrocyte responses, comparable to those obtained with Th HA cell culture supernatants containing IBF alpha. These studies show that Th HA cells express Fc alpha R with less Fc mu R, and the solubilized form of Fc alpha R exhibits IBF alpha-like activity. The significance of FcR expression by Th cell clones and cell lines and the relationship of soluble Fc alpha R and IBF alpha for IgA response regulation are discussed.     

T cell hybridomas coexpressing Fc receptors (FcR) for different isotypes. II. IgA-induced formation of suppressive IgA binding factor(s) by a murine T hybridoma bearing Fc gamma R and Fc alpha R

T2D4 murine T hybridoma cells have previously been shown to express Fc receptors (FcR) for IgG (Fc gamma R) and for IgA (Fc alpha R) and to produce an IgG binding factor (IgGBF) that suppresses IgG and IgM responses. In the present work we report on the behavior of IgA bound to T2D4 cells and on the production of IgA binding factor (IgABF) and its ability to suppress IgA antibody production. A dose-dependent binding of MOPC315 IgA with anti-TNP activity by T2D4 cells was demonstrated by rosette formation with trinitrophenylated ox red blood cells (TNP-ORBC) and fixation of iodinated DNP-BSA. IgA bound to the cells disappeared after a short-term culture of 3 hr at 37 degrees C, but not at 4 degrees C. Because this phenomenon was inhibited by 0.1% sodium azide and 100 microM dansylcadaverine, a transglutaminase inhibitor, Fc alpha R-IgA complexes seemed to be released by an active process involving receptor movement. In the culture supernatant of IgA-treated T2D4 cells, we detected a factor(s) that binds to IgA-Sepharose and competitively inhibits the binding of IgA to T2D4 cells. The factor (IgABF) failed to inhibit the rosette formation of Fc gamma R(+) cells with IgG-sensitized ORBC (EAox gamma), indicating that it binds specifically to IgA. IgABF was undetectable in the culture supernatants of untreated T2D4 cells of Fc alpha R(-) BW5147 T lymphoma cells used as parent cells for the establishment of the hybridoma. To study the effect of IgABF on antibody formation, culture filtrates of IgA-treated or untreated T2D4 cells were fractionated on IgA-Sepharose beads and were added to BALB/c spleen cells cultured with pokeweed mitogen. By use of a reverse plaque assay, it was shown that the IgA plaque-forming cell (PFC) response was suppressed by the acid eluate but not by the effluent of IgA-Sepharose beads incubated with the filtrates of IgA-treated T2D4 cell cultures. The suppression was IgA specific, because neither IgG nor IgM responses were suppressed by the eluate. As expected, there was no significant IgA suppressive activity in the acid eluates of the beads incubated with the culture filtrate of untreated T2D4 cells or IgA-treated BW5147 cells. IgA-specific suppressive activity proved to be due to IgA binding factor(s), because suppressive activity in the eluate was completely adsorbed by IgA-Sepharose but not by IgG- nor BSA-Sepharose.     

Nonhuman primate IgA: genetic heterogeneity and interactions with CD89

Nonhuman primates are extremely valuable animal models for a variety of human diseases. However, it is now becoming evident that these models, although widely used, are still uncharacterized. The major role that nonhuman primate species play in AIDS research as well as in the testing of Ab-based therapeutics requires the full characterization of structure and function of their Ab molecules. IgA is the Ab class mostly involved in protection at mucosal surfaces. By binding to its specific Fc receptor CD89, IgA plays additional and poorly understood roles in immunity. Therefore, Ig heavy alpha (IGHA) constant (C) genes were cloned and sequenced in four different species (rhesus macaques, pig-tailed macaques, baboons, and sooty mangabeys). Sequence analysis confirmed the high degree of intraspecies polymorphism present in nonhuman primates. Individual animals were either homozygous or heterozygous for IGHA genes. Highly variable hinge regions were shared by animals of different geographic origins and were present in different combinations in heterozygous animals. Therefore, it appears that although highly heterogeneous, hinge sequences are present only in limited numbers in various nonhuman primate populations. A macaque recombinant IgA molecule was generated and used to assess its interaction with a recombinant macaque CD89. Macaque CD89 was able to bind its native ligand as well as human IgA1 and IgA2. Presence of Ag enhanced macaque IgA binding and blocking of macaque CD89 N-glycosylation reduced CD89 expression. Together, our results suggest that, despite the presence of IgA polymorphism, nonhuman primates appear suitable for studies that involve the IgA/CD89 system.     

Enhancement of antibody-dependent cell-mediated cytotoxicity by endowing IgG with FcαRI (CD89) binding

Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are crucial to the efficacy of many antibody therapeutics. In addition to IgG, antibodies of the IgA isotype can also promote cell killing through engagement of myeloid lineage cells via interactions between the IgA-Fc and FcαRI (CD89). Herein, we describe a unique, tandem IgG1/IgA2 antibody format in the context of a trastuzumab variable domain that exhibits enhanced ADCC and ADCP capabilities. The IgG1/IgA2 tandem Fc format retains IgG1 FcγR binding as well as FcRn-mediated serum persistence, yet is augmented with myeloid cell-mediated effector functions via FcαRI/IgA Fc interactions. In this work, we demonstrate anti-human epidermal growth factor receptor-2 antibodies with the unique tandem IgG1/IgA2 Fc can better recruit and engage cytotoxic polymorphonuclear (PMN) cells than either the parental IgG1 or IgA2. Pharmacokinetics of IgG1/IgA2 in BALB/c mice are similar to the parental IgG, and far surpass the poor serum persistence of IgA2. The IgG1/IgA2 format is expressed at similar levels and with similar thermal stability to IgG1, and can be purified via standard protein A chromatography. The tandem IgG1/IgA2 format could potentially augment IgG-based immunotherapeutics with enhanced PMN-mediated cytotoxicity while avoiding many of the problems associated with developing IgAs.     

Antisera to the secretory component recognize the murine Fc receptor for IgA

Studies were undertaken to determine a possible structural relationship between the secretory component (SC) and the receptor for IgA (Fc alpha R). An IgA-mediated rosetting technique was used to assess the presence of Fc alpha R+ cells in various lymphoid tissues from normal BALB/c mice and mice bearing an IgA plasmacytoma (MOPC 315). Tissues from the MOPC 315-bearing BALB/c mice were found to have a significantly higher percentage of Fc alpha R+ cells; thus, nonadherent spleen cells from MOPC 315-bearing mice were used as a source of Fc alpha R+ cells in these studies. The cells were preincubated with anti-SC and then assayed for the ability of IgA to bind to the Fc alpha R. Antisera to SC from various species inhibited the formation of IgA-mediated rosettes, although preincubation of the Fc alpha R+ cells with antisera directed against other cell surface molecules (e.g., Thy1.2, Lyt1, Lyt2, Fc gamma R, MHC class I and II) or preimmune sera had no significant effect on IgA-mediated rosette formation. Preabsorption of the anti-SC with secretory IgA or with free SC removed the inhibitory effect; preabsorption with myeloma IgA had no effect. These data suggest that SC and Fc alpha R are related serologically and may be structurally related, possible in the IgA-binding region.