Human Igs Research Articles

Use of monoclonal antibodies against horse immunoglobulin in an enzyme immunoassay of bacterial toxins and toxoids

Immunization of BALB/c mice by horse antiserum against diphtheria made it possible to obtain IgG1 monoclonal antibodies (MoAbs) 2B7E4 specific for light chains of horse immunoglobulin (Ig). Unlike commercial preparations of anti-horse immunoglobulin antibodies, which are specific for the whole Ig molecule or its Fc-fragment, the peroxidase (HRP) conjugate of the MoAb, 2B7E4-HRP did not interact with human, mouse, rabbit, and sheep Igs, or horse albumin. The conjugate obtained was used with MoAbs against bacterial toxins and commercial horse anatoxins, as a universal reagent in sandwich enzyme immunoassay (ELISA) for bacterial toxins and anatoxins. The detection sensitivity of diphtheria toxin/anatoxin equaled 0.0005 Lf/ml; tetanus toxin and anatoxin were detected with sensitivities of 20 LD50/ml and 0.005 UI/ml, respectively. A similar sandwich ELISA for botulinum anatoxins (group measurement) allowed types A, B, and E to be detected at 0.02, 0.002, and 0.001 UI/ml, respectively; selective measurement was only possible in the case of type E anatoxin (0.001 UI/ml).

B Cell Superantigen-induced Immune Complex Lung Inflammation

RATIONALE: Peptostreptococcus magnus Protein L functions as a B cell superantigen (SAg) by binding to Vκappa light chain determinants on many human and mouse Igs, irrespective of their antigen specificities. The ability of B cell SAgs to bind to a large amount of serum IgG underscores their potential to cause immune complex-mediated tissue injury. We investigated whether such a B cell SAg elicits inflammation when introduced into the airways and, if so, whether the reaction resembles that elicited by immune complexes. METHODS: C57BL/6 mice were administered either Protein L or BSA intratracheally (i.t.). Bronchoalveolar lavage fluid (BALF) was collected at various time points and analyzed for total neutrophil counts. Simultaneously obtained lung samples were analyzed for myeloperoxidase (MPO) and histology. Studies were repeated in mast cell deficient mice and wildtype littermates to investigate potential contributions of mast cells. RESULTS: Protein L-treated mice accumulated neutrophils in their BALF peaking at 24 hours after i.t. challenge. Their lungs showed margination of leukocytes in blood vessel lumens and a perivascular infiltrate of inflammatory cells. These changes were accompanied by diapedesis of erythrocytes and, in some areas, frank hemorrhage. They were associated with markedly increased tissue concentrations of MPO and were preserved in mast cell deficient mice. CONCLUSIONS: A B cell SAg can induce mast cell-independent inflammation in the lungs with features suggestive of immune complex mediated injury. Since most B cell SAgs are derived from ubiquitous microbes, these results may help elucidate how infectious organisms contribute to the pathogenesis of immune complex mediated lung injury.

Degalactosylated and/or Denatured IgA, but Not Native IgA in Any Form, Bind to Mannose-Binding Lectin

Mannose-binding lectin (MBL) is reported to bind to agalactosyl IgG, but not to normally galactosylated (native) IgG. It was recently reported that serum polymeric IgA in its native form reacts with MBL, whereas a more recent report has claimed that native IgD and IgE, and possibly IgM, do not. This led us to investigate whether IgA is truly reactive with MBL. To accomplish this, we collected purified human Igs, of various classes, subclasses, and allotypes, and tested their ability to bind to MBL using an ELISA method. Among these preparations, only one (monoclonal IgA2m(2):Kur) exhibited significant MBL binding. In particular, polymeric or monomeric forms of our normal serum IgA preparation lacked any ability to bind to MBL whatsoever. However, all the Ig preparations which had not bound to MBL became able to do so when they were degalactosylated with a galactosidase treatment, and the binding was further enhanced by acidic denaturation of the Igs. Among the degalactosylated and/or acid-denatured IgA, the IgA2 subclass exhibited a higher level of MBL binding than did IgA1. Our results suggest that MBL does not bind to native Igs (viewed in principle as "self" components), and that only Igs with abnormal glycosylation (degalactosylated forms) and/or denaturation would be MBL reactive.

FcRn mediates elongated serum half-life of human IgG in cattle

IgG has the longest survival time in the circulation of the Ig classes and the lowest fractional catabolic rate. The neonatal Fc receptor (FcRn) plays an important role in regulating these processes. Recently, we have cloned the bovine neonatal Fc receptor (bFcRn) alpha chain and detected its expression in various epithelial cells which are mediating IgG secretion. However, its function in IgG homeostasis has not been investigated. In the current study, we analyzed the binding affinity of bovine and human IgGs to bFcRn using surface plasmon resonance and by in vitro radioreceptor binding assays. As human IgG binds stronger to the bFcRn, than bovine IgG at pH 6, we subsequently analyzed its catabolism in normal and transchromosomic calves that produce human Igs. Pharmacokinetic studies showed that human IgG had approximately 33 days serum half-life both in normal and transchromosomic calves, which is more than two times longer than its bovine counterpart. We also demonstrate FcRn expression in endothelial cells and in the kidney which are supposed to be involved in IgG metabolism. These data suggest that bFcRn is involved in IgG homeostasis in cattle and furthermore, that the transchromosomic calves producing human Igs can effectively protect their human IgGs which have implications for successful large-scale production of therapeutic antibodies.

Cross-reaction of anti-simian immunodeficiency virus envelope protein antibodies with human immunoglobulins.

It has been recently established that retroviral envelope proteins (REPs) have structural features similar to those of immunoglobulins (Igs). In this study, we asked whether anti-REP antibodies cross-react with human Igs (hIgs). To this end, murine monoclonal antibodies (mMoAbs) that had been raised against a simian immunodeficiency virus (SIV) envelope protein, SIVMac251gp120, were screened for their ability to react with human monoclonal Igs (HMIgs). We show that two HMIgs, RFSJ2 (a rheumatoid factor) and PAMLN6 (a human anti-hIg V region antibody), but not a number of other HMIgs, could be weakly, but consistently, bound by anti-SIVMac251gp120 mMoAbs KK17 and KK46, as judged by indirect enzyme-linked immunosorbent assay and a liquid-phase inhibition immunoassay. Both mMoAbs are specific to amino acid residues in the V3 loop of the SIVMac251gp120. The RFSJ2 Ig heavy-chain V region (VH) is coded in part by a human VH gene, VH3-30.3 and includes the idiotope 7B4 (NKYY), which was previously shown to be present in the gp120 protein of a number of HIV-2 and SIV strains. However, an entirely different VH gene codes the PAMLN6 VH region, opening the possibility that epitope(s) shared between SIVMac251gp120 and hIgs may not be limited to the 7B4 idiotope.

Tracking immunoglobulin variable-gene expression in HIV infection.

B-cell superantigens (SAgs) interact with normal human nonimmune immunoglobulins (Igs) independently of the light-chain isotype, and activate a large proportion of the B-cell repertoire. Recently, the major envelope protein of human immunodeficiency virus 1 (HIV-1), gp120, was found to exhibit SAg-like properties for B cells with potential pathological consequences for the infected host, including accelerated apoptosis and progressive loss of B cells. This unconventional mode of interaction contrasts with its binding to immunization-induced antibodies, which requires the tertiary structure of the heavy- and light-chain variable regions. Examining the temporal development of V(H)3+ antibodies in HIV-1-infected subjects over a 7-yr period showed that V(H)3+ antibodies specific for the gp120 SAg-binding site are deficient. Quantification of V(H)3+ antibodies, which impart protective responses to infectious agents, in serum samples from HIV-seropositive slow progressors and from patients who progressed to AIDS-related manifestations reveals that paucity in V(H)3+ antibodies is a marker of rapid clinical decline. Remarkably, anti-gp160 V(H)3+ antibodies show a gradual decrease in progressors and vary with time, depending on the viral load. Thus, V(H)3+ antibodies could play an important role in protection, and their underexpression may accelerate disease progression. Investigation of the structural basis of the interaction between human Igs and gp120 shows that the viral gp120 SAg can interact only with a subset of human V(H)3+ Igs. A number of amino acid-positions present primarily in the first and third framework regions of the Ig heavy-chain variable regions correlate with gp120 binding. These residues partially overlap with the Staphylococcus aureus protein A-binding site for V(H)3+ Igs. Overall, these interactions could represent a novel mechanism of humoral deficiency resulting from the capacity of a viral SAg to impact an important subset of the B-cell repertoire and to induce B-cell death by apoptosis.

Structural Basis of the gp120 Superantigen-Binding Site on Human Immunoglobulins

B cell superantigens (SAg) interact with normal human nonimmune Igs (Igs), independently of the light chain isotype, and activate a large proportion of the B cell repertoire. Recently, the major envelope protein of HIV-1, gp120, was found to exhibit SAg-like properties for B cells with potential pathologic consequences for the infected host. This unconventional mode of interaction contrasts with its binding to immunization-induced Abs, which requires the tertiary structure of the heavy and light chain variable regions. In this report, we have examined the structural basis of the interaction between human Igs and gp120. We found that gp120 binding is restricted to Igs from the V(H)3 gene family and that the two V(H) genes 3-23 and 3-30, known to be overutilized during all stages of B cell development, frequently impart gp120 binding. We also provide evidence that the viral gp120 SAg can interact with only a subset of the human V(H)3+ Igs that can convey binding to the prototypic bacterial B cell SAg protein A from Staphylococcus aureus. Finally, we have identified amino acid positions present primarily in the first and third framework regions of the Ig heavy chain variable region, outside the conventional hypervariable loops, which correlate with gp120 binding. In a three-dimensional sequence-homology model, these residues partially overlap with the predicted SAg protein A binding site for V(H)3+ Igs.

Primary Myeloma Cells Growing in SCID-hu Mice: A Model for Studying the Biology and Treatment of Myeloma and Its Manifestations

Progress in unraveling the biology of myeloma has suffered from lack of an in vitro or in vivo system for reproducible growth of myeloma cells and development of disease manifestations. The SCID-hu mouse harbors a human microenvironment in the form of human fetal bone. Myeloma cells from the bone marrow of 80% of patients readily grew in the human environment of SCID-hu mice. Engraftment of myeloma cells was followed by detectable human Ig levels in the murine blood. Myeloma-bearing mice had high levels of monotypic human Igs, high blood calcium levels, increased osteoclast activity, and severe resorption of the human bones. The human microenvironment was infiltrated with Epstein-Barr virus-negative monoclonal myeloma cells of the same clonality as the original myeloma cells. Active angiogenesis was apparent in areas of myeloma cell infiltration; the new endothelial cells were of human origin. We conclude that the SCID-hu mouse is a favorable host for studying the biology and therapy of myeloma and that a normal bone marrow environment can support the growth of myeloma cells. © 1998 by The American Society of Hematology.

Primary Myeloma Cells Growing in SCID-hu Mice: A Model for Studying the Biology and Treatment of Myeloma and Its Manifestations

Progress in unraveling the biology of myeloma has suffered from lack of an in vitro or in vivo system for reproducible growth of myeloma cells and development of disease manifestations. The SCID-hu mouse harbors a human microenvironment in the form of human fetal bone. Myeloma cells from the bone marrow of 80% of patients readily grew in the human environment of SCID-hu mice. Engraftment of myeloma cells was followed by detectable human Ig levels in the murine blood. Myeloma-bearing mice had high levels of monotypic human Igs, high blood calcium levels, increased osteoclast activity, and severe resorption of the human bones. The human microenvironment was infiltrated with Epstein-Barr virus-negative monoclonal myeloma cells of the same clonality as the original myeloma cells. Active angiogenesis was apparent in areas of myeloma cell infiltration; the new endothelial cells were of human origin. We conclude that the SCID-hu mouse is a favorable host for studying the biology and therapy of myeloma and that a normal bone marrow environment can support the growth of myeloma cells.© 1998 by The American Society of Hematology.

In Vivo Inflammatory Response to a Prototypic B Cell Superantigen: Elicitation of an Arthus Reaction by Staphylococcal Protein A

Staphylococcal protein A (SpA) is representative of a new class of Ags, the B cell superantigens (SAgs). These SAgs, unlike conventional Ags, bind to the Fab regions of Ig molecules outside their complementarity-determining regions. In addition, B cell SAgs can react with a substantial amount of a host's serum Igs by virtue of their ability to interact with many members of an entire variable heavy chain (VH) or variable light chain gene family. For example, SpA reacts with the Fabs of most human Igs using heavy chains from the VH3 gene family (VH3+). Members of this gene family are expressed on 30 to 60% of human peripheral B cells. We sought to determine whether the interaction of a B cell SAg with its reactive Igs can elicit immune complex-mediated tissue injury. Using the Arthus reaction in rabbits as an in vivo model of immune complex-mediated tissue inflammation, we demonstrated that untreated rabbits, which were administered SpA intradermally (i.d.), do not develop a cutaneous inflammatory response. However, when rabbits were pretreated i.v. with human IgG (hIgG), i.d. injections of SpA induced an inflammatory response with the classical histologic features of an Arthus reaction. To determine whether this Arthus-like response occurred via a B cell superantigenic mechanism, the rabbits were pretreated with VH3-depleted hIgG and then were administered SpA i.d. We found that the induction of a prominent inflammatory response by SpA was dependent upon the presence of VH3+ molecules in the hIgG pretreatment. These results provide compelling evidence that an interaction of the B cell SAg, SpA, with its reactive (VH3+) IgGs leads to an immune complex-mediated inflammatory response in vivo.

Induction of interleukin-1 and subsequent tissue factor expression by anti-proteinase 3 antibodies in human umbilical vein endothelial cells.

To assess the ability of anti-proteinase 3 (anti-PR3) classic antineutrophil cytoplasmic antibodies (cANCA) to stimulate endothelial expression of tissue factor (TF), which is the main initiator of the coagulation cascade that can lead to endothelial injury and thrombosis in patients with Wegener's granulomatosis. Human umbilical vein endothelial cells (HUVEC) were grown to confluence and stimulated with affinity-purified anti-PR3 antibodies, Igs from healthy subjects, and endotoxin (lipopolysaccharide) as positive control. TF activity was generated in anti-PR3-stimulated cells, as shown by a chromogenic test. This activity was inhibited by specific anti-TF antibodies. TF messenger RNA (mRNA) was found in anti-PR3-stimulated cells, as detected by reverse transcriptase-polymerase chain reaction, but not in cells stimulated with irrelevant human Igs or Igs from normal control sera. TF expression reached maximum levels 12 hours after exposure to the anti-PR3 cANCA, and did not require complement. TF mRNA expression was inhibited by cycloheximide, suggesting a requirement for protein synthesis. When added to the incubation medium, interleukin-1 (IL-1) receptor antagonist inhibited the induced TF mRNA expression, suggesting that cANCA-stimulated cells initiate IL-1 synthesis. Moreover, cANCA induced IL-1alpha mRNA before TF mRNA. This study showed that anti-PR3 treatment of HUVEC induces sequential expression of IL-1alpha mRNA and TF mRNA, as well as their corresponding proteins. Both proteins could have pathogenic roles in the vasculitic process, since TF is the main initiator of the coagulation cascade.

Identification of the B-cell superantigen binding site of HIV-1 gp120

Previous studies showed that the gp120 envelope protein of HIV-1 is able to crosslink membrane IgM on normal human B cells and to induce their activation in a VH3 immunoglobulin gene-family-specific manner. Because this VH gene family is the largest in the human repertoire, this superantigen (SAg) property is thought to have deleterious consequences for the host, including a progressive decline of B cells with progression of the HIV-1-induced disease. Here, we have identified the sequence motifs on gp120 involved in SAg binding to normal Igs. We show that this SAg-binding activity is present in gp120s from highly divergent isolates of HIV-1 belonging to clades derived from various geographical origins, and that carbohydrate residues are not essential for its expression. The SAg-binding site is formed by protein sequences from two regions of the gp120 molecule. The core motif is a discontinuous epitope spanning the V4 variable domain and the amino-terminal region flanking the C4 constant domain. The most critical residues appear to be Leu395–Asp397 and Ile425–Gln427. Residues from the C2 constant domain (positions 252–272) also seem to play an accessory role in SAg binding of gp120 to normal human Igs. These findings are important in the design of a successful gp120-based vaccine against HIV-1.

A Model for Human B-Chronic Lymphocytic Leukemia in Human/Mouse Radiation Chimera: Evidence for Tumor-Mediated Suppression of Antibody Production in Low-Stage Disease

AbstractB-chronic lymphocytic leukemia (BCLL) is a lymphoproliferative disease that is characterized by clonal expansion of CD5+ B cells. BCLL is associated with secondary immunodeficiency and hypogammaglobulinemia. It has been suggested that T-cell dysregulation may play a role in the hypogammaglobulinemia and in the increased incidence of autoimmunity in BCLL patients. We attempted to transfer human peripheral blood mononuclear cells (PBMC) from BCLL patients in different stages of the disease into immunodeficient mice. PBMC from BCLL patients in stage 0, stages I to II, and stages III to IV were transplanted into the peritoneal cavity of lethally irradiated Balb/c or beige/nude/Xid (BNX) mice radioprotected with bone marrow (BM) from severe combined immunodeficiency (SCID) mice. Different engraftment profiles were found in the chimeric mice 2 weeks after transplantation of PBMC according to the disease stage of the BCLL donors. Infusion of PBMC from donors in stage 0 led to marked engraftment of human T cells, whereas the human tumor cells could hardly be detected. In contrast, chimeric mice receiving PBMC from patients in stage III to IV disease exhibited engraftment with a dominance of tumor cells, compared with a miniscule level of T cells. The ability of the engrafted cells to produce human Ig was also found to be correlated with the disease stage of the donor, although all donors had the same magnitude of hypogammaglobulinemia. Total human Ig production in the chimeric mice was normal in mice receiving PBMC from donors in stage 0, whereas in chimeric mice engrafted with PBMC from donors in stages III to IV almost no human Igs could be detected. This differential reconstitution of antibody production in the mouse model according to the stage of the patient's disease will allow further studies on possible cellular interactions between malignant and immune cells in BCLL.

An endogenous sialoprotein and a bacterial B cell superantigen compete in their VH family-specific binding interactions with human Igs.

Staphylococcal protein A (SpA), a bacterial membrane protein, and protein Fv (Fv binding protein (pFv)), a human sialoprotein involved in gut-associated immunity, have both recently been shown to have unconventional V(H) family-restricted binding interactions with Igs. To determine whether these Ig binding proteins interact with related structures, we performed a series of comparative binding studies. The results confirmed that both molecules are bound by most V(H)3 IgM, but pFv is also recognized by V(H)3 and V(H)6 Ig that do not interact with SpA. We discovered that pFv and SpA (or a single domain of SpA) can compete for binding to a V(H)3 Ab, which suggests that they can recognize the same (or adjacent) V(H) sites. For both SpA and pFv, binding is less frequent among IgG than IgM. However, V(H)3 IgG more commonly possess Fab-mediated binding activity for pFv than for SpA. Binding studies of denatured Ig suggested that both pFv and SpA interact with conformationally dependent V(H) sites, although in certain cases pFv binding is more permissive than SpA binding. Taken together, these results indicate that the superantigen properties of SpA, a microbial protein, and those of pFv, an endogenous sialoprotein, involve binding interactions with overlapping and at times functionally equivalent sites in the V(H) domain, indicating that self and foreign proteins can employ highly conserved strategies to create superantigens for the Ag receptors of B lymphocytes.

Can immunoglobulin C(H)1 constant region domain modulate antigen binding affinity of antibodies?

Although the switch process is frequently associated with affinity maturation, the constant region is not assumed to play a role in Ag-Ab binding. In the present work, we demonstrate that two clonally related human monoclonal Igs sharing identical V(H) and V(L) sequences, but expressing different isotypes (IgA1kappa(PER) and IgG1kappa(PER)), bind tubulin with significantly different affinities. This difference was mainly accounted for by a disparity in the association rate constants. These results suggest that affinity maturation of this clone could be achieved through class switching in the absence of further somatic mutations. Since the differences observed were found at the Fab level, they also suggest a role for the C(H)1 domain in structuring the Ag-binding site into a more kinetically competent form.

Staphylococcal protein A simultaneously interacts with framework region 1, complementarity-determining region 2, and framework region 3 on human VH3-encoded Igs.

Staphylococcal protein A (SPA) is a B cell superantigen that binds to human VH3-encoded Igs independently of the D- and JH-encoded regions and light chain sequences. The SPA-binding structure formed by VH3-encoded Igs remains controversial. We localized the regions in a VH3-encoded Ab required for SPA binding by producing mutant Abs in the baculovirus expression system in which regions of a human-derived Ab known to bind SPA were exchanged with those from a mouse Ab of the J558 family, a family not associated with SPA binding. The pattern of SPA binding indicates not only that residues in FR1, CDR2, and FR3 are involved but also that the three regions are required to interact simultaneously with SPA for binding to occur. When any one of the three regions was replaced with the corresponding region from the nonbinding Ab, SPA binding was severely disrupted. These data indicate that SPA requires simultaneous interaction with three distinct regions of a VH3 structure, which together in three-dimensional space form an extended solvent-exposed surface. These studies more precisely define the genetic requirements for VH3-encoded Ig binding to SPA.

Human Immunoglobulins Produced in hu-PBL-SCID Mice Are Polyclonal Early after Xenotransplantation

Xenotransplantation of human peripheral blood leukocytes (hu-PBL) to severe combined immune-deficient (SCID, hu-PBL-SCID) mice leads to human immunoglobulin (Ig) production in the animals. Here we used a sensitive high-resolution two-dimensional electrophoresis (2D-PAGE) technique to separate, and to analyze, Ig γ and light chains in sera from SCID mice given hu-PBL for less than 35 days. Human Igs in the model appeared polyclonal until 3 weeks after cell transfer. After this period, oligoclonal Igs progressively replaced this polyclonal background. The 2D-PAGE pattern evolution of human Igs was similar when hu-PBL originated from donors with or without previous contact with the Epstein–Barr virus. The broad diversity of human Igs, and therefore the large expressed B cell repertoire, in hu-PBL-SCID mice during a period that allows manipulation (such as immunization) of the human graft has evident implication for the use of this model in the generation of specific antibodies of desired specificity.

Analysis of human immunoglobulin-degrading cysteine proteinases of Trichomonas vaginalis

Trichomonas vaginalis is a protozoan parasite that causes a widely distributed sexually transmitted disease (STD). Since immunoglobulin G (IgG) antibodies to specific trichomonad immunogens are found in serum and vaginal washes (VWs) from patients with trichomoniasis, a potential mechanism of immune evasion by this parasite might be the ability of T. vaginalis proteinases to degrade human immunoglobulins (Igs). Incubation of human IgG with lysates of T. vaginalis organisms resulted in time- and concentration-dependent degradation of the heavy chain. Secretory IgA was degraded similarly. Inhibitors of cysteine proteinases, when added to trichomonal lysates, abolished IgG and IgA degradation, while EDTA, a metalloproteinase inhibitor, did not. Substrate-gel electrophoresis with human IgG, IgM, or IgA copolymerized with acrylamide revealed several distinct cysteine proteinases in both lysates and culture supernatants from logarithmically growing parasites that degraded all classes of human antibodies. Trichomonal lysates and supernatants of numerous isolates tested all had Ig-degrading activity. Finally, proteolytic activity against IgG was detected in most (26 of 33; 78%) VWs from patients with trichomoniasis. In contrast, 18 of 28 (65%) VWs from women without trichomoniasis or from patients infected with other STDs had no detectable proteinases when tested in an identical manner. The other 10 of these 28 VWs (35%) had smaller amounts of detectable Ig-degrading proteinases. These differences in Ig-degrading proteinase activity between patients with and without trichomoniasis, regardless of coinfecting STDs, were statistically significant (P = 0.001). These results illustrate that T. vaginalis is capable of degrading human Igs.

Enzymatically mediated, glycosidic conjugation of immunoglobulins with viral epitopes

We developed a novel enzymatic procedure to couple a peptide to the sugar moieties of immunoglobulins (Igs). The synthesis of the conjugates consists in galactose (Gal) oxidation of desialylated Igs followed by covalent attachment of the peptides with concurrent stabilization of the Schiff bases upon mild reduction. The peptide used in this study, corresponds to the amino acid residues 110–120 of hemagglutinin (HA) of PR8 A virus and is recognized by CD4 T helper cells in association with I-E d class II major histocompatibility complex (MHC). The degree of coupling as determined by competitive inhibition of radioimmunoassay (IRIA) using FPLC purified conjugates was estimated at 11.4 peptides per IgG molecule. Coupling of HA110–120 peptide to the sugar moiety of various mouse and human Igs was confirmed by Western blot analysis developed with anti-HA110–120 antibodies. Complete detachment of the peptide from the conjugates by N-deglycosylation with PGNase F indicated a defined specificity of coupling HA peptide to the N-linked oligosaccharides of Igs. To facilitate quick release of the peptides from the conjugates into the lysosomal compartment of the antigen processing cells (APC) we introduced at the α amino terminus of the peptide (HAc110–120), a cleavage site for cathepsins (AAAL). The immunoglobulin-galactose-HAc110–120 conjugates (IGP) were able to activate HA110–120 specific T hybridoma cells as efficient as influenza PR8 A virus and 40–100-fold higher than the synthetic peptide itself.

Human immunoglobulin (IgG) induced deletion of IgM rheumatoid factor B cells in transgenic mice.

The singular ability of immunoglobulin genes to hypermutate their variable regions, while permitting the generation of high-affinity antibodies against foreign antigens, poses a problem in terms of maintenance of immunological self-tolerance. Immunoglobulin gene hypermutation driven by a foreign antigen has the potential to generate antibodies that cross-react with self-components. Consequently, there must exist a mechanism in the periphery for inactivation of mature autoreactive B cell clones. The classical experimental system used to address this problem is the induction of tolerance to soluble, deaggregated human IgG. We have analyzed the mechanism of induction of tolerance to human IgG using transgenic mice that express a human IgM rheumatoid factor (IgM RF) on a large proportion of their B cells. Injection of deaggregated human IgG caused a specific deletion of those B cells that express an intact IgM RF on their cell surface. The degree of RF B cell deletion was proportional to the reduction in the proliferative response of splenocytes to antigen (aggregated human IgG), or to F(ab')2 fragments of anti-human IgM antibodies. Control experiments showed that IgG administration had little effect on the numbers of mouse Ig-bearing cells or their ability to proliferate to a nonspecific mitogen. Thus, the effects of IgG on the human IgM RF B cell are antigen specific and are not due to nonspecific toxic effects of the human IgG preparation. These experiments demonstrate that peripheral exposure to IgG induces deletion of reactive B cells, without any evidence for anergy, and differ from data obtained by other investigators studying tolerance to soluble protein antigens. The results imply that human Igs have distinct properties as soluble antigens, and that peripheral nonresponsiveness to IgG may be due to lymphocyte deletion.