The IgA/IgM receptor expressed on a murine B cell lymphoma is poly-Ig receptor

T560, a mouse B lymphoma that originated in gut-associated lymphoid tissue, expresses receptors that bind dimeric IgA and IgM in a mutually inhibitory manner but have little affinity for monomeric IgA. Evidence presented in this paper indicates that the receptor is poly-Ig receptor (pIgR) known in humans and domestic cattle to bind both IgA and IgM. The evidence includes the demonstration that binding of IgM is J chain dependent, and that pIg-precipitated receptor has an appropriate Mr of 116-120 kDa and can be detected on immunoblots with specific rabbit anti-mouse pIgR. Overlapping RT-PCR performed using template mRNA from T560 cells and oligonucleotide primer pairs designed from the published sequence of mouse liver pIgR indicate that T560 cells express mRNA virtually identical with that of the epithelial cell pIgR throughout its external, transmembrane, and intracytoplasmic coding regions. Studies using mutant IgAs suggest that the Calpha2 domain of dimeric IgA is not involved in high-affinity binding to the T560 pIgR. Inasmuch as this mouse B cell pIgR binds IgM better than IgA, it is similar to human pIgR and differs from rat, mouse, and rabbit epithelial cell pIgRs that bind IgA but not IgM. Possible explanations for this difference are discussed. All clones of T560 contain some cells that spontaneously secrete both IgG2a and IgA, but all of the IgA recoverable from the medium and from cell lysates is monomeric; it cannot be converted to secretory IgA by T560 cells.     

Synergistic effect of IL-4 and IFN-gamma on the expression of polymeric Ig receptor (secretory component) and IgA binding by human epithelial cells

The expression of secretory component (SC), the epithelial receptor for polymeric Ig, was enhanced by the addition of human rIFN-gamma or rIL-4, as revealed by the binding of radiolabeled polymeric, J chain-containing IgA or anti-SC antisera to the human colonic adenocarcinoma epithelial cell line HT-29. In combination, these cytokines exhibited a synergistic effect, and the potentiating effect of IL-4 was inhibitable by polyclonal anti-IL-4 antisera. Because the binding of radiolabeled polymeric IgA (pIgA) to HT-29 cells was inhibited by unlabeled pIgA or a polyclonal anti-SC reagent, but not by IgG, monomeric IgA, or Fab alpha fragments, we conclude that the receptor involved in the increased binding of pIgA is indeed SC. These data suggest that the expression of SC on human epithelial cells and the subsequent binding of pIgA (produced in mucosal tissues and glands by subepithelial plasma cells) is regulated by lymphokines such as IL-4 and IFN-gamma that are presumably derived from T cells found in abundant numbers in these tissues. These findings demonstrate a novel pathway of interaction between T cell products and epithelial cells that may result in enhanced translocation of large amounts of locally produced pIgA through epithelial cells into external secretions.     

Studies of surface immunoglobulins on human B lymphocytes. I. Dissociation of cell-bound immunoglobulins with acid pH or at 37 degrees C.

Lymphocyte preparations isolated from the human peripheral blood were exposed to different acid pH or incubated at 37 degrees C and the presence of immunoglobulin (Ig) on the cell surface was examined by immunofluorescence (IF) tests. Subsequently, such treated cells were incubated in the autologous serum or in the purified IgG, IgA or IgM proteins and their ability to bind each class of Ig was examined. The results showed that IgG molecules dissociated from large proportions of IgG-positive cells upon exposure to pH 4 at 1 degrees C for 1 min or upon incubation at 37 degrees C for 20 min. The cells from which IgG had been dissociated could again combine with IgG, whereupon the number of positive cells increased, being restored to the number of equivalent to or higher than those before acid or 37 degrees C treatment. These results indicated that the treatment could elute the cell-bound IgG present on the cell and that the receptor sites were not degraded by the treatment and could combine with IgG. These cell-bound IgG were observed not only on the monocytes, but also on the small lymphocytes. It was also found that certain proportions of mononuclear cells carried the cell-bound IgA that could be dissociated with acid pH or 37 degrees C. No cell-bound IgM was observed on any mononuclear cells. Microscopic observations before and after acid or 37 degrees C treatment revealed that the staining distribution of the cell-bound IgG and IgA on the cell was granular, appearing as a discontinuous fluorescence ring and forming multiple aggregates but no typical polar caps on warming. In contrast, IgG, IgA, and IgM stable to acid or 37 degrees C treatment were found on the lymphocytes but not on the monocytes, and their staining distribution was uniformaly diffuse, appearing as a continuous ring and forming a typical cap on warming. Exposure of the cells to pH 4 or 37 degrees C could also elute the cell-bound IgG passively adsorbed to the human lymphoid cells in a culture, but did not affect the intrinsic S.Ig on the lymphoid cells in a culture or on the lymphoma cells. These results indicate that the exposure of the cells to acid pH or to 37 degrees C may enable us to detect unfailingly S.Ig lymphocytes by removing the cell-bound IgG and IgA present on the monocytes and/or lymphocytes. Thus, an average value of approximately 10% was obtained for the S.Ig lymphocyte in the lymphocyte preparations from 11 healthy individuals. In addition, the results provided the evidence that, even in normal peripheral blood lymphocytes, there may be a population of B lymphocytes which lack the S.Ig but carry the cell-bound Ig.     

A role for isotype-specific binding factors in the regulation of IgA- and IgG-specific responses by the anti/contrasuppressor T cell circuit

Previous studies have shown that the isotype of an antibody response is selected, in part, by the inhibition of isotype-specific suppression. The antisuppressor model predicts that isotype selection is initiated through an interaction between Ag, Ig, and a T cell-derived factor within 6 h of immunization. This report characterizes some of these molecules and their contribution to isotype regulation. Cultures of murine spleen cells stimulated with the T cell-dependent Ag SRBC led to Ag-specific IgG and IgA responses that could be suppressed and then antisuppressed by a molecular complex produced by mixing purified serum Ig with the supernatant of Ag-pulsed macrophages co-cultured with T cells. The supernatants from separate cultures of Ag-pulsed macrophages and rIL-1 alpha stimulated CD4+ T cells, could be pooled and mixed with Ig to produce functional antisuppressive complexes thereby allowing the factors from the different cell types to be studied separately. Adsorption of the co-culture or the rIL-1 alpha stimulated T cell supernatants against monoclonal IgG or IgA, removed IgG and IgA binding factors, respectively, and abrogated the ability to enhance the corresponding isotype. The adherent material could be recovered and used to reconstitute enhancement by the supernatants depleted of the binding factors. When affinity purified IgG or IgA was used as the source of Ig within the antisuppressive complexes, the enhancement of the antibody response was limited to the isotype of the regulatory Ig used to form the complex. Thus, manipulation of the antisuppressive molecules has a predictable effect on isotype selection. Release of isotype-specific binding factors by CD4+ cells by rIL-1 alpha supports the hypothesis that T cell circuits play a role in initiating isotype regulation.     

In vitro and in vivo activation of murine gamma/delta T cells induces the expression of IgA, IgM, and IgG Fc receptors.

The present studies examined resting and activated murine gamma/delta T lymphocytes, in vitro and in vivo, for surface expression of FcR. Polyclonal gamma/delta TCR+ lymphocytes selectively grown from the spleen and intestine of normal mice did not express FcR when the cells were in a resting state, but when cells were activated with anti-CD3 antibody virtually all of the splenic gamma/delta lymphocytes and a large subpopulation of the intestinal gamma/delta lymphocytes expressed IgA and IgM FcR. This was confirmed by using transgenic mice. Resting gamma/delta TCR+ lymphocytes from the spleen, thymus, lymph node, and blood of gamma/delta TCR transgenic mice did not express FcR for any of the five major classes of Ig H chains. Activation of the gamma/delta TCR+ cells via the CD3/TCR complex induced high levels of IgM and IgA FcR and low levels of IgG FcR. Finally, in hepatic granulomas of schistosome-infected mice, activated gamma/delta TCR+ cells are present and express high levels of IgA and IgM FcR and low levels of IgG FcR. These investigations establish that transition of gamma/delta TCR+ lymphocytes from a resting to an activated state (triggered via the T3Ti TCR complex) is accompanied by the induction of surface membrane receptors specific for Ig H chain isotypes. The activation-linked expression of FcR on gamma/delta TCR+ lymphocytes provides potential mechanisms for coupling the functional activities of gamma/delta T lymphocytes with immune mechanisms that involve Ig molecules, such as antibody-dependent cellular cytotoxicity.     

Intracellular neutralization of influenza virus by immunoglobulin A anti-hemagglutinin monoclonal antibodies.

Traditionally, immunoglobulin A (IgA) was thought to neutralize virus by forming complexes with viral attachment proteins, blocking attachment of virions to host epithelial cells. Recently we have proposed an intracellular action for dimeric IgA, which is actively transported through epithelial cells by the polymeric immunoglobulin receptor (pIgR), in that it may be able to bind to newly synthesized viral proteins within the cell, preventing viral assembly. To this effect, we have previously demonstrated that IgA monoclonal antibodies against Sendai virus, a parainfluenza virus, colocalize with the viral hemagglutinin-neuraminidase protein within infected epithelial cells and reduce intracellular viral titers. Here we determine whether IgA can interact with influenza virus hemagglutinin (HA) protein within epithelial cells. Polarized monolayers of Madin-Darby canine kidney epithelial cells expressing the pIgR were infected on their apical surfaces with influenza virus A/Puerto Rico/8-Mount Sinai. Polymeric IgA anti-HA, but not IgG anti-HA, delivered to the basolateral surface colocalized with HA protein within the cell by immunofluorescence. Compared with those of controls, viral titers were reduced in the supernatants and cell lysates from monolayers treated with anti-HA IgA but not with anti-HA IgG. Furthermore, the addition of anti-IgA antibodies to supernatants did not interfere with the neutralizing activity of IgA placed in the basal chamber, indicating that IgA was acting within the cell and not in the extracellular medium to interrupt viral replication. Thus, these studies provide additional support for the concept that IgA can inhibit replication of microbial pathogens intracellularly.     

IgA-induced avidity maturation of IgA Fc receptors on murine T lymphocytes

The analysis of 30 well characterized murine T lymphocyte populations using a cytofluorometric IgA binding assay has identified many populations that are constitutive and/or inducible for IgA receptor expression, and has identified two distinct mechanisms by which IgA up-regulates the IgA-binding properties of murine T cells. Studies with lymphomas, hybridomas, Ag-specific clones and activated normal splenic T cells identified many examples of CD4 and CD8 lineage cells that constitutively expressed IgA receptors. T cell populations that constitutively expressed IgA receptors exhibited enhanced IgA binding after incubation with oligomeric IgA for 18 h. The IgA-induced up-regulation of IgA binding resulted from two distinct processes: 1) an increase in the number of surface membrane IgA binding sites and 2) an increase in the avidity of IgA binding without a change in the number of binding sites. The IgA-induced avidity increase was reflected by a 5- to 10-fold decrease in the apparent Kd. Depending on the T cell population examined the enhanced binding of IgA involved one or both of these mechanisms. T cell populations that did not constitutively express IgA receptors failed to bind IgA after prolonged incubation with oligomeric IgA suggesting that if such cells can express IgA receptors they require other signals to induce their expression. Consistent with this possibility is the finding that resting splenic T cells did not bind IgA but their activation with Con A or mAb anti-T3 resulted in high level expression of IgA receptors. These studies have identified multiple distinct mechanisms that alter the IgA-binding properties of murine T cells and are discussed in terms of their possible physiologic significance.     

Human intestinal epithelial cells express a novel receptor for IgA

Binding and transport of polymeric Igs (pIgA and IgM) across epithelia is mediated by the polymeric Ig receptor (pIgR), which is expressed on the basolateral surface of secretory epithelial cells. Although an Fc receptor for IgA (FcalphaR) has been identified on myeloid cells and some cultured mesangial cells, the expression of an FcalphaR on epithelial cells has not been described. In this study, binding of IgA to a human epithelial line, HT-29/19A, with features of differentiated colonic epithelial cells, was examined. Radiolabeled monomeric IgA (mIgA) showed a dose-dependent, saturable, and cation-independent binding to confluent monolayers of HT-29/19A cells. Excess of unlabeled mIgA, but not IgG or IgM, competed for the mIgA binding, indicating that the binding was IgA isotype-specific and was not mediated by the pIgR. The lack of competition by asialoorosomucoid and the lack of requirement for divalent cations excluded the possibility that IgA binding to HT-29/19A cells was due to the asialoglycoprotein receptor or beta-1, 4-galactosyltransferase, previously described on HT-29 cells. Moreover, the FcalphaR (CD89) protein and message were undetectable in HT-29/19A cells. FACS analysis of IgA binding demonstrated two discrete populations of HT-29/19 cells, which bound different amounts of mIgA. IgA binding to other colon carcinoma cell lines was also demonstrated by FACS analysis, suggesting that an IgA receptor, distinct from the pIgR, asialoglycoprotein receptor, galactosyltransferase, and CD89 is constitutively expressed on cultured human enterocytes. The function of this novel IgA receptor in mucosal immunity remains to be elucidated.     

Comparative studies on FcR (FcRII, FcRIII, and FcR alpha) functions of murine B cells

Distribution of FcR II, FcRIII, and FcR alpha on murine splenic B cells was examined by using FITC-labeled heat-aggregated IgG of each subclass and IgA. Almost 60 to 80% of B cells expressed both FcRII and FcRIII. However, FcR alpha was expressed on only a small proportion (6%) of B cells that co-expressed FcRII. By inhibition assays with the use of cold IgG of each subclass and IgA in addition to anti-FcRII mAb (2.4G2), it was found that IgG1, IgG2a, and IgG2b utilized the same receptor (FcRII), whereas IgG3 and IgA bound only to their unique receptors, FcRIII and FcR alpha, respectively. Immune complexes IC prepared by IgG1, IgG2a, IgG2b, and IgA anti-TNP mAb with TNP-coupled SRBC inhibited the polyclonal Ig secretion and proliferative responses of B cells stimulated with either IL-4 or LPS. The inhibition of B cell activation was associated with the blockade of the membrane depolarization. Moreover, IC prepared by these antibodies caused production of suppressive B cell factor (SBF) as is the case with rabbit IgG antibody to SRBC, and SBF thus prepared regulated antibody responses in an isotype-nonspecific manner. In contrast, no inhibition for these responses or production of SBF was attained by the IC of IgG3 antibody. We concluded that FcRII and FcR alpha mediates a suppressive signal for B cells by acting on the initial step of activation, whereas FcRIII lacks this activity.     

Synthetic Fc peptide-mediated regulation of the immune response. II. Analysis of secreted immunoglobulin classes, accessory cell contribution, and lymphocyte proliferation in p23-stimulated spleen cell cultures

The synthetic peptide p23 representing residues 335 to 357 in the CH3 domain of human IgG1 was able to increase levels of secreted Ig in murine spleen cell cultures. This in vitro response was optimal in the presence of between 10(-4) and 10(-3) micron p23/ml and the levels of secreted Ig reached a maximum on day 4 or day 5 of culture. Supernatants from p23-treated cell cultures generally contained more IgM than IgG and undetectable levels of IgA. Induction of Ig secretion by p23 was macrophage-independent but T cell-dependent and, with respect to the latter case, removal of T cells from spleen cells reduced the levels of both IgM and IgG. Although maintaining the B cell differentiation-inducing quality of its progenitor molecule, the Fc gamma fragment, p23 appeared to have lost the ability to induce B cell proliferation. Evidence is presented that a sequence functionally similar to p23 is extant in mouse IgG by showing that murine Fc gamma fragments were also able to induce increases in Ig-secreting cells in murine spleen cell cultures.     

Dimerization of the Polymeric Immunoglobulin Receptor Controls Its Transcytotic Trafficking

Binding of dimeric immunoglobulin (Ig)A to the polymeric Ig receptor (pIgR) stimulates transcytosis of pIgR across epithelial cells. Through the generation of a series of pIgR chimeric constructs, we have tested the ability of ligand to promote receptor dimerization and the subsequent role of receptor dimerization on its intracellular trafficking. Using the cytoplasmic domain of the T cell receptor-ζ chain as a sensitive indicator of receptor oligomerization, we show that a pIgR:ζ chimeric receptor expressed in Jurkat cells initiates a ζ-specific signal transduction cascade when exposed to dimeric or tetrameric IgA, but not when exposed to monomeric IgA. In addition, we replaced the pIgR’s transmembrane domain with that of glycophorin A to force dimerization or with a mutant glycophorin transmembrane domain to prevent dimerization. Forcing dimerization stimulated transcytosis of the chimera, whereas preventing dimerization abolished ligand-stimulated transcytosis. We conclude that binding of dimeric IgA to the pIgR induces its dimerization and that this dimerization is necessary and sufficient to stimulate pIgR transcytosis.     

Alternative endocytic pathway for immunoglobulin A Fc receptors (CD89) depends on the lack of FcRgamma association and protects against degradation of bound ligand

IgA is the most abundant immunoglobulin in mucosal areas but is only the second most common antibody isotype in serum because it is catabolized faster than IgG. IgA exists in monomeric and polymeric forms that function through receptors expressed on effector cells. Here, we show that IgA Fc receptor(s) (FcalphaR) are expressed with or without the gamma chain on monocytes and neutrophils. gamma-less FcalphaR represent a significant fraction of surface FcalphaR molecules even on cells overexpressing the gamma chain. The FcalphaR-gamma2 association is up-regulated by phorbol esters and interferon-gamma. To characterize gamma-less FcalphaR functionally, we generated mast cell transfectants expressing wild-type human FcalphaR or a receptor with a point mutation (Arg --> Leu at position 209) which was unable to associate with the gamma chain. Mutant gamma-less FcalphaR bound monomeric and polymeric human IgA1 or IgA2 but failed to induce exocytosis after receptor clustering. The two types of transfectant showed similar kinetics of FcalphaR-mediated endocytosis; however, the endocytosis pathways of the two types of receptor differed. Whereas mutant FcalphaR were localized mainly in early endosomes, those containing FcalphaR-gamma2 were found in endo-lysosomal compartments. Mutant gamma-less FcalphaR recycled the internalized IgA toward the cell surface and protected against IgA degradation. Cells expressing the two forms of FcalphaR, associated or unassociated with gamma chains, may thus have differential functions either by degrading IgA antibody complexes or by recycling serum IgA.     

IgM complex receptors on subpopulations of murine lymphocytes.

Murine lymphocytes from spleen, lymph node, and thymus were examined for IgM complex receptors. Lymphocytes from all three organs were found to bind SRBC sensitized with IgM from various sources including: primary anti-SRBC serum, murine and rabbit anti-Escherichia coli LPS sera, and a murine IgM myeloma (MOPC 104E). Rosette formation by lymphocytes with IgM-sensitized SRBC was inhibited by soluble antigen-IgM complexes but not by IgM or antigen alone. Rosette formation was also inhibited by human IgM (Fc)5mu but not by Fab mu. Antiserum and complement treatment of the cells and subsequent recovery of the viable cells by trypsinization, filtration, and washing revealed the IgM rosette-forming cell (RFC) in the thymus to be a T cell. Spleen on the other hand was found to contain both B and T cells capable of binding IgM sensitized SRBC. Removal of both B and T cells from spleen cell suspensions eliminated all IgM RFC. The IgM complex receptor was found to be trypsin insensitive. Anti-Ig column fractionation enriched IgM RFC in spleen and lymph node suspensions passed through the columns, whereas cells bearing surface Ig, IgG complex receptors, and C3 receptors were retained in the columns.     

Induction of exosome release in primary B cells stimulated via CD40 and the IL-4 receptor

Exosomes are lipid-bound nanovesicles formed by inward budding of the endosomal membrane and released following fusion of the endosomal limiting membrane with the plasma membrane. We show here that primary leukocytes do not release exosomes unless subjected to potent activation signals, such as cytokine or mitogen stimulation. In particular, high levels of exosomes were released when murine splenic B cells were stimulated via CD40 and the IL-4 receptor. This property was shared by B cells from different anatomic locations, as newly formed marginal zone and follicular B cells were capable of secreting exosomes upon CD40/IL-4 triggering. B cell exosomes expressed high levels of MHC class I, MHC class II, and CD45RA (B220), as well as components of the BCR complex, namely, surface Ig, CD19, and the tetraspanins CD9 and CD81. Ig on the plasma membrane of primary B cells was targeted to the exosome pathway, demonstrating a link between the BCR and this exocytic pathway. IgD and IgM were the predominant Ig isotypes associated with CD40/IL-4 elicited exosomes, though other isotypes (IgA, IgG1, IgG2a/2b, and IgG3) were also detected. Together, these results suggest that exosome release is not constitutive activity of B cells, but may be induced following cell: cell signaling.     

Expression, distribution and specificity of Fc receptors for IgM on murine B cells

Subpopulations of normal adult murine splenic B cells and a panel of murine B cell tumors were examined for their ability to bind murine IgM specifically. By using two-color flow cytometric analyses, we have demonstrated that 90 to 95% of surface (s)IgD+ B cells express surface membrane receptors for IgM (Fc mu R). The binding of pentameric murine IgM to splenocyte Fc mu R was IgM-specific since it was totally inhibited by other polymeric IgM proteins, but not by Ig of other H chain classes or by mAb specific for the murine IgG or IgE FcR. Binding of IgM to splenic cells was saturable. Fc mu R were co-expressed with the Fc gamma R as well as the Fc epsilon R on the majority of splenic B cells. Minor populations of splenic mononuclear cells expressed only an Fc mu R, Fc gamma R or Fc epsilon R. In a survey of B tumor cell lines representing different stages of B cell development, we observed that the Fc mu R was expressed on pre-B cell lines and that Fc mu R detection was maximal on immature B cell lines that expressed sIgM and low amounts of sIgD and Ia. Fc mu R were not detected on cell lines that had switched from sIgM to the expression of another sIg, or on plasmacytomas and hybridomas. The studies with normal splenocytes establish that the majority of sIgD+ B lymphocytes in adult BALB/c mice express surface membrane receptors that specifically bind IgM. The studies with B lineage tumor cells suggest that the expression of Fc mu R on B cells is developmentally regulated and that the pattern of expression exhibited by Fc mu R during B cell ontogeny differs from the patterns that have been previously found for IgG and IgE FcR. These observations raise the possibility that Fc mu R might have a functional significance in some aspect of B cell maturation and activation. By using a family of IgM H chain constant region domain deletional mutants, we have further demonstrated that, like the T cell Fc mu R, the B cell Fc mu R also requires a C mu 3 domain for binding to occur, raising the possibility that the T and B cell Fc mu R in mice may be structurally related molecules.     

Intraepithelial cell neutralization of HIV-1 replication by IgA

HIV is transmitted sexually through mucosal surfaces where IgA Abs are the first line of immune defense. In this study, we used paired IgA and IgG mAbs against HIV gp160 to study intraepithelial cell neutralization and inhibition of HIV replication. African green monkey kidney cells, Vero C1008, polarizable epithelial cells transfected to express the polymeric Ig receptor (pIgR), were transfected with HIV proviral DNA, and intracellular neutralization mediated by the mAbs was assessed. D47A and D19A IgA, which neutralized HIV in a conventional assay, potently inhibited intracellular HIV replication as assessed by infecting HeLa-CD4-long terminal repeat/beta-galactosidase cells (human cervical carcinoma cell line) and CEMx174 cells (human T cell line) with apical supernatant, basolateral medium, and cell lysate from transfected cells. D47A also inhibited the production of virus as assessed by direct assay of p24. In contrast, D47 and D19 IgG, sharing the same V regions, but which were not transcytosed by the pIgR, did not inhibit intracellular HIV replication, nor did D47A and D19A IgA in pIgR- cells, incapable of transcytosing IgA. Confocal immunofluorescence microscopy showed prominent colocalization of HIV protein and D47A, in agreement with the intracellular neutralization data. D10A, which did not neutralize HIV in the conventional assay, and irrelevant IgA did not show intracellular neutralization or colocalization. Control studies with two kinds of conditioned medium confirmed that HIV neutralization had indeed occurred inside the cells. Thus, during its transcytosis through epithelial cells, HIV-specific IgA can neutralize HIV replication.     

Oligosaccharide side chains on human secretory IgA serve as receptors for ricin

Secretory IgA (sIgA) Abs are polymeric Igs comprised of two or more IgA monomers joined together at their C termini and covalently associated with a 70-kDa glycoprotein called secretory component. As the predominant Ig type in gastrointestinal sections, sIgA Abs are centrally important in adaptive immunity to enteropathogenic bacteria, viruses, and toxins. In this study, we demonstrate that sIgA Abs may also function in innate defense against ricin, a naturally occurring, galactose-specific plant lectin with extremely potent shiga toxin-like enzymatic activity. In lectin blot overlay assays, we found that ricin bound to secretory component and the H chain of human IgA, and this binding was inhibited by the addition of excess galactose. The toxin also recognized IgM (albeit with less affinity than to IgA), but not IgG. Ricin bound to both human IgA1 and IgA2, primarily via N-linked oligosaccharide side chains. At 100-fold molar excess concentration, sIgA (but not IgG) Abs inhibited ricin attachment to the apical surfaces of polarized intestinal epithelial cells grown in culture. sIgA Abs also visibly reduced toxin binding to the luminal surfaces of human duodenum in tissue section overlay assays. We conclude that sIgA Abs in mucosal secretions may serve as receptor analogues for ricin, thereby reducing the effective dose of toxin capable of gaining access to glycolipid and glycoprotein receptors on epithelial cell surfaces.     

Recombinant dimeric IgA antibodies against the epidermal growth factor receptor mediate effective tumor cell killing

Dimeric IgA Abs contribute significantly to the humoral part of the mucosal immune system. However, their potential as immunotherapeutic agent has hardly been explored. In this article, we describe the production, purification, and functional evaluation of recombinant dimeric IgA against the epidermal growth factor receptor. Human joining chain-containing IgA was produced by nonadherent Chinese hamster ovarian (CHO)-K1 cells under serum-free conditions. Purification by anti-human κ and anti-His-tag affinity, as well as size exclusion chromatography, resulted in a homogenous preparation of highly pure IgA dimers. Functional studies demonstrated dimeric IgA to be at least as effective as monomeric IgA in triggering Ab-dependent cellular cytotoxicity by isolated monocytes or polymorphonuclear cell and in human whole-blood assays. Importantly, dimeric IgA was more effective in F(ab)-mediated killing mechanisms, such as inhibition of ligand binding, receptor downmodulation, and growth inhibition. Furthermore, only dimeric but not monomeric IgA or IgG was directionally transported by the polymeric Ig receptor through an epithelial cell monolayer. Together, these studies demonstrate that recombinant dimeric IgA Abs recruit a distinct repertoire of effector functions compared with monomeric IgA or IgG1 Abs.     

Fc-receptors for IgG and IgM immunoglobulins on human T lymphocytes: mode of re-expression after proteolysis or interaction with immune complexes.

The susceptibility to proteolysis and the mode of re-expression of receptors for IgM or IgG present on two different subpopulations of human T lymphocytes (T.M and T.G cells, respectively) have been investigated. The IgM receptor was highly susceptible to both trypsin and pronase, whereas the IgG receptor was resistant to trypsin and sensitive only to high concentrations of pronase. The receptors have been removed by treating purified human T cells with pronase and their reappearance on the cell surface has been followed in vitro. The IgM receptors on the cell surface were detectable within 2 hr and the resynthesis was completed in 6 hr. IgG receptors were detectable in 4 to 6 hr and the resynthesis completed within 12 hr. When protein synthesis was inhibited by culturing the cells in the presence of cycloheximide for up to 12 hr, only the IgM receptor (which had a higher turnover rate) failed to be expressed. Whereas interaction of IgG immune complex with the IgG receptors was previously shown to induce a modulation of the receptors, contact with antigen-IgM antibody complexes did not alter the mode of expression of IgM receptors.     

Lymphocyte function in human bone marrow. I. Characterization of two T cell populations regulating immunoglobulin secretion

We analyzed the regulation of immunoglobulin (Ig) production in short-term cultures of human (rib) bone marrow cells. In contrast to blood or tonsil cell cultures, large quantities of IgG and IgA, but not IgM, were secreted by unstimulated marrow cells. The addition of pokeweed mitogen or phytohemagglutinin resulted in the suppression of this Ig secretion. Both mitogens induced the production of high levels of interleukin 2 (IL 2) in marrow cultures, and the addition of IL 2 alone mimicked the suppressive effect of mitogens. Incubation of marrow cells with Epstein Barr virus resulted in enhanced Ig secretion, primarily of the IgM isotype. The addition of mitogen or IL 2 suppressed Ig production in these cultures as well. The mitogen-induced suppression of Ig secretion in stimulated or unstimulated marrow cultures was inhibited by the monoclonal anti-TAC (IL 2 receptor) antibody. Cell separation experiments indicated that the induction of suppressor activity in marrow cultures involved two distinct populations of marrow-resident T lineage cells. The first population responds to activation by mitogens with the production of IL 2. This population has a surface phenotype appropriate for helper T cells. The second T cell population expresses T8 and TAC determinants. These cells acquire suppressor cell activity after exposure to IL 2. The expression of suppressor function does not require additional (e.g., mitogenic) activation signals. The IL 2-dependent marrow suppressor T cells represent a newly recognized T lymphocyte subset. The regulatory pathway delineated may be important for the regulation of antibody formation in bone marrow, the major site of Ig production in man.