Immunoglobulin G3 from polyclonal human immunodeficiency virus (HIV) immune globulin is more potent than other subclasses in neutralizing HIV type 1.

Passive antibody prophylaxis against human immunodeficiency virus type 1 (HIV-1) has been accomplished in primates, suggesting that this strategy may prove useful in humans. While antibody specificity is crucial for neutralization, other antibody characteristics, such as subclass, have not been explored. Our objective was to compare the efficiencies of immunoglobulin G (IgG) subclasses from polyclonal human HIV immune globulin (HIVIG) in the neutralization of HIV-1 strains differing in coreceptor tropism. IgG1, IgG2, and IgG3 were enriched from HIVIG by using protein A-Sepharose. All three subclasses bound major HIV-1 proteins, as shown by Western blot assay and enzyme-linked immunosorbent assay. In HIV-1 fusion assays using X4, R5, or X4R5 envelope-expressing effector cells, IgG3 more efficiently blocked fusion. In neutralization assays with cell-free viruses using X4 (LAI, IIIB), R5 (BaL), and X4R5 (DH123), a similar hierarchy of neutralization was found: IgG3 > IgG1 > IgG2. IgG3 has a longer, more flexible hinge region than the other subclasses. To test whether this is important, IgG1 and IgG3 were digested with pepsin to generate F(ab')(2) fragments or with papain to generate Fab fragments. IgG3 F(ab')(2) fragments were still more efficient in neutralization than F(ab')(2) of IgG1. However, Fab fragments of IgG3 and IgG1 demonstrated equivalent neutralization capacities and the IgG3 advantage was lost. These results suggest that the IgG3 hinge region confers enhanced HIV-neutralizing ability. Enrichment and stabilization of IgG3 may therefore lead to improved HIVIG preparations. The results of this study have implications for the improvement of passive immunization with polyclonal or monoclonal antibodies and suggest that HIV-1 vaccines which induce high-titer IgG3 responses could be advantageous.     

The generation and evaluation of two panels of epitope-matched mouse IgG1, IgG2a, IgG2b and IgG3 antibodies specific for Plasmodium falciparum and Plasmodium yoelii merozoite surface protein 1-19 (MSP1(19))

Murine immunoglobulin G (IgG) plays an important role in mediating protective immune responses to malaria. We still know relatively little about which IgG subclasses protect against this disease in mouse models, although IgG2a and IgG2b are considered to be the most potent and dominate in successful passive transfer experiments in rodent malarias. To explore the mechanism(s) by which the different mouse IgG subclasses may mediate a protective effect, we generated mouse IgG1, IgG2a, IgG2b and IgG3 specific for the C-terminal 19-kDa region of Plasmodium falciparum merozoite surface protein 1 (PfMSP1(19)), and to the homologous antigen from Plasmodium yoelii (P. yoelii), both major targets of protective immune responses. This panel of eight IgGs bound antigen with an affinity comparable to that seen for their epitope-matched parental monoclonal antibodies (mAbs) from which they were derived, although for reasons of yield, we were only able to explore the function of mouse IgG1 recognizing PfMSP1(19) in detail, both in vitro and in vivo. Murine IgG1 was as effective as the parental human IgG from which it was derived at inducing NADPH-mediated oxidative bursts and degranulation from neutrophils. Despite showing efficacy in in vitro functional assays with neutrophils, the mouse IgG1 failed to protect against parasite challenge in vivo. The lack of protection afforded by MSP1(19)-specific IgG1 against parasite challenge in wild type mice suggests that this Ab class does not play a major role in the control of infection with mouse malaria in the Plasmodium berghei transgenic model.     

Poorly neutralizing cross-reactive antibodies against the fusion loop of West Nile virus envelope protein protect in vivo via Fcgamma receptor and complement-dependent effector mechanisms

The human antibody response to flavivirus infection is dominantly directed against a cross-reactive epitope on the fusion loop of domain II (DII-FL) of the envelope (E) protein. Although antibodies against this epitope fail to recognize fully mature West Nile virus (WNV) virions and accordingly neutralize infection poorly in vitro, their functional properties in vivo remain less well understood. Here, we show that while passive transfer of poorly neutralizing monoclonal antibodies (MAb) and polyclonal antibodies against the DII-FL epitope protect against lethal WNV infection in wild-type mice, they fail to protect mice lacking activating Fcγ receptors (FcγR) and the complement opsonin C1q. Consistent with this, an aglycosyl chimeric mouse-human DII-FL MAb (E28) variant that lacks the ability to engage FcγR and C1q also did not protect against WNV infection in wild-type mice. Using a series of immunodeficient mice and antibody depletions of individual immune cell populations, we demonstrate that the nonneutralizing DII-FL MAb E28 does not require T, B, or NK cells, inflammatory monocytes, or neutrophils for protection. Rather, E28 treatment decreased viral load in the serum early in the course of infection, which resulted in blunted dissemination to the brain, an effect that required phagocytic cells, C1q, and FcγRIII (CD16). Overall, these studies enhance our understanding of the functional significance of immunodominant, poorly neutralizing antibodies in the polyclonal human anti-flavivirus response and highlight the limitations of current in vitro surrogate markers of protection, such as cell-based neutralization assays, which cannot account for the beneficial effects conferred by these antibodies.     

CD16 Expression on Monocytes in Healthy Individuals but Not Schistosome-Infected Patients Is Positively Associated with Levels of Parasite-Specific IgG and IgG1

Human IgG1 antibody responses are associated with protection against Schistosoma haematobium infection and are now a target for schistosome vaccine development. This study aimed to investigate the relationship between total IgG and the IgG subclasses and the monocyte IgG receptor, known as FcγRIIIa or CD16, in schistosome exposed people. Systemic levels of schistosome-specific anti-adult worm total IgG and IgG subclass titres were measured by ELISA in 100 individuals from an S. haematobium endemic area in Zimbabwe and, using parametric statistical methods and regression analysis, related to the levels of CD16 expression on individuals'' circulating monocytes, determined via flow cytometry. Monocyte CD16 expression rose with parasite-specific total IgG and IgG1 in healthy participants, but not in schistosome infected patients. Similar to parasite-specific IgG and IgG1, CD16 expression in healthy individuals is associated with protection against schistosome infection. This relationship indicates a mechanistic link between the innate and adaptive immune responses to helminth infection in protection against infection. Further understanding the elements of a protective immune response in schistosomiasis may aid in efforts to develop a protective vaccine against this disease.     

Central role of complement in passive protection by human IgG1 and IgG2 anti-pneumococcal antibodies in mice

Streptococcus pneumoniae is an important cause of morbitity and mortality worldwide. Capsule-specific IgG1 and IgG2 Abs are induced upon vaccination with polysaccharide-based vaccines that mediate host protection. We compared the protective capacity of human recombinant serogroup 6-specific IgG1 and IgG2 Abs in mice deficient for either leukocyte FcR or complement factors. Human IgG1 was found to interact with mouse leukocyte FcR in vitro, whereas human IgG2 did not. Both subclasses induced complement activation, resulting in C3c deposition on pneumococcal surfaces. Passive immunization of C57BL/6 mice with either subclass before intranasal challenge with serotype 6A induced similar degrees of protection. FcgammaRI- and III-deficient mice, as well as the combined FcgammaRI, II, and III knockout mice, were protected by passive immunization, indicating FcR not to be essential for protection. C1q or C2/factor B knockout mice, however, were not protected by passive immunization. Passively immunized C2/factor B(-/-) mice displayed higher bacteremic load than C1q(-/-) mice, supporting an important protective role of the alternative complement pathway. Spleens from wild-type and C1q(-/-) mice showed hyperemia and thrombotic vessel occlusion, as a result of septicemic shock. Notably, thrombus formation was absent in spleens of C2/factor B(-/-) mice, suggesting that the alternative complement pathway contributes to shock-induced intravascular coagulation. These studies demonstrate complement to play a central role in Ab-mediated protection against pneumococcal infection in vivo, as well as in bacteremia-associated thrombotic complications.     

Th1-type immune response to a Coccidioides immitis antigen delivered by an attenuated strain of the non-invasive enteropathogen Vibrio cholerae

The antigen-2 or proline rich antigen (Ag2/PRA) from Coccidioides immitis, known to protect mice against experimental Coccidioidomycosis, was expressed in the genetically attenuated cholera vaccine candidate Vibrio cholerae 638 and its thymine auxotrophic derivative 638T. Intranasal immunization of mice with strains producing Ag2/PRA induced serum vibriocidal antibody and Ag2/PRA-specific total IgG responses in outbred Swiss Webster and inbred BALB/c mice. Analysis of IgG subclasses showed a predominance of IgG2a subclass antibodies. Lymphocytes from immunized mice stimulated with pure Ag2/PRA showed a significant proliferative response with production of interferon-gamma. Positive selection for plasmid maintenance in vivo did not enhance immune response to Ag2/PRA. These results demonstrate that genetically attenuated strains of the non-invasive pathogen V. cholerae can be used to express and deliver foreign antigens to stimulate a Th1 type of immune response.     

Vaccinia Virus Extracellular Enveloped Virion Neutralization In Vitro and Protection In Vivo Depend on Complement

Antibody neutralization is an important component of protective immunity against vaccinia virus (VACV). Two distinct virion forms, mature virion and enveloped virion (MV and EV, respectively), possess separate functions and nonoverlapping immunological properties. In this study we examined the mechanics of EV neutralization, focusing on EV protein B5 (also called B5R). We show that neutralization of EV is predominantly complement dependent. From a panel of high-affinity anti-B5 monoclonal antibodies (MAbs), the only potent neutralizer in vitro (90% at 535 ng/ml) was an immunoglobulin G2a (IgG2a), and neutralization was complement mediated. This MAb was the most protective in vivo against lethal intranasal VACV challenge. Further studies demonstrated that in vivo depletion of complement caused a >50% loss of anti-B5 IgG2a protection, directly establishing the importance of complement for protection against the EV form. However, the mechanism of protection is not sterilizing immunity via elimination of the inoculum as the viral inoculum consisted of a purified MV form. The prevention of illness in vivo indicated rapid control of infection. We further demonstrate that antibody-mediated killing of VACV-infected cells expressing surface B5 is a second protective mechanism provided by complement-fixing anti-B5 IgG. Cell killing was very efficient, and this effector function was highly isotype specific. These results indicate that anti-B5 antibody-directed cell lysis via complement is a powerful mechanism for clearance of infected cells, keeping poxvirus-infected cells from being invisible to humoral immune responses. These findings highlight the importance of multiple mechanisms of antibody-mediated protection against VACV and point to key immunobiological differences between MVs and EVs that impact the outcome of infection.     

Malaria resistance genes are associated with the levels of IgG subclasses directed against Plasmodium falciparum blood-stage antigens in Burkina Faso

ABSTRACT: BACKGROUND: HBB, IL4, IL12, TNF, LTA, NCR3 and FCGR2A polymorphisms have been associated with malaria resistance in humans, whereas cytophilic immunoglobulin G (IgG) antibodies are thought to play a critical role in immune protection against asexual blood stages of the parasite. Furthermore, HBB, IL4, TNF, and FCGR2A have been associated with both malaria resistance and IgG levels. This suggests that some malaria resistance genes influence the levels of IgG subclass antibodies. METHODS: In this study, the effect of HBB, IL4, IL12, TNF, LTA, NCR3 and FCGR2A polymorphisms on the levels of IgG responses against Plasmodium falciparum blood-stage extract was investigated in 220 individuals living in Burkina Faso. The Pearson's correlation coefficient among IgG subclasses was determined. A family-based approach was used to assess the association of polymorphisms with anti-P. falciparum IgG, IgG1, IgG2, IgG3 and IgG4 levels. RESULTS: After applying a multiple test correction, several polymorphisms were associated with IgG subclass or IgG levels. There was an association of i) haemoglobin C with IgG levels; ii) the FcgammaRIIa H/R131 with IgG2 and IgG3 levels; iii) TNF-863 with IgG3 levels; iv) TNF-857 with IgG levels; and, v) TNF1304 with IgG3, IgG4, and IgG levels. CONCLUSION: Taken together, the results support the hypothesis that some polymorphisms affect malaria resistance through their effect on the acquired immune response, and pave the way towards further comprehension of genetic control of an individual's humoral response against malaria.     

The IgE and IgG subclass responses of mice to four helminth parasites

To investigate whether the formation of IgE is linked in vivo to an IgG subclass, mice were infected with four helminth parasites, Nippostrongylus brasiliensis (Nbr), Mesocestoides corti, Taenia crassiceps and Trichinella spiralis, and the changes in the serum levels of the different Ig isotypes as well as the antibody response to M. corti and T. crassiceps antigen extracts were determined by radioimmunoassays. All four parasites induced a concomitant increase of the IgE and IgG1 serum levels and usually a decrease of the IgG2a level. They also induced an increase of the IgM level but had little effect on the IgG2b, IgG3, and IgA serum levels. The specific antibodies to an M. corti antigen extract were mainly of the IgG1 subclass, whereas it was of both IgG1 and IgG2a subclasses to T. crassiceps. Injections of dead M. corti induced an increase of all IgG subclasses and similar levels of IgG1 and IgG2a anti-parasite antibodies. Subcutaneous instead of intraperitoneal infection with T. crassiceps induced higher IgG2a than IgG1 levels and 10-fold lower IgE levels than the natural ip infection; however, despite the greater IgG2a polyclonal response, anti-parasite antibodies were predominantly of the IgG1 subclass. The data demonstrate that natural infection with four different helminth parasites induces a concomitant polyclonal IgG1 and IgE response. These in vivo observations corroborate the recent in vitro findings demonstrating that interleukin-4 induces lipopolysaccharide-activated murine B cells to secrete both IgG1 and IgE, suggesting that the regulation of these two isotypes is linked.     

IgG subclass expression by human B lymphocytes and plasma cells: B lymphocytes precommitted to IgG subclass can be preferentially induced by polyclonal mitogens with T cell help

The human IgG subclasses expressed by circulating B lymphocytes, tissue plasma cells, and plasma cells generated from B cell precursors in response to the polyclonal mitogens LPS and PWM were examined by immunofluorescence using subclass-specific monoclonal antibodies. The subclass distribution observed for circulating B lymphocytes was IgG2 (48%) greater than IgG1 (40%) greater than IgG3 (8%) greater than IgG4 (1%), while the distribution among IgG plasma cells in bone marrow, blood, spleen, and tonsils was IgG1 (64%) greater than IgG2 (26%) greater than IgG3 (8%) greater than IgG4 (1%). Multiple IgG isotypes were not observed on B cells or in plasma cells. Although IgG plasma cell responses to both LPS and PWM were T cell dependent, the distributions of IgG subclasses elicited were strikingly different. In control and LPS-stimulated cultures of blood mononuclear cells, the induced plasma cells expressed the IgG subclass distribution: IgG2 greater than 80%, IgG1 less than 20%, IgG3 less than 1%, IgG4 less than 1%. In PWM-stimulated cultures, the subclass distribution, IgG1 approximately 65%, IgG2 approximately 25%, IgG3 approximately 7%, IgG4 approximately 1%, was in perfect concordance with the in vivo subclass distribution of IgG plasma cells. Selective inhibition of suppressor T cell activity by x-irradiation and mitomycin C treatment did not alter the IgG subclass distribution pattern induced by LPS and PWM. Monoclonal antibodies were used to deplete selectively the B cell precursors bearing IgG1, IgG2, or IgG3 before PWM stimulation of blood mononuclear cells. In each instance, a reduction was observed only in the subpopulation of plasma cells producing the homologous IgG subclass. The results indicate that T cells can preferentially influence the terminal differentiation of B cells that are precommitted to different IgG subclasses.     

Trypanosoma cruzi: Role of different antibody classes in protection against infection in the mouse

Immune serum obtained from mice with a chronic infection of Trypanosoma cruzi was fractionated on Sephadex G-200 or on protein ASepharose 4B. Mice were infected with a standard infective dose of T. cruzi 24 hr after injection with either IgM, IgG, IgG1, or IgG2 + IgG3 fractions. Mice were also pretreated with immune serum depleted by affinity chromatography of either IgG2a, IgG2b, or both subclasses before infection with T. cruzi. Control mice were pretreated with normal mouse serum or immune serum depleted of IgG. The parasitemia and survival of the animals were determined and used as parameters of protection. The results of these experiments demonstrated that the protective antibodies were mostly IgG2 and seem to be preferentially located in IgG2b subclass. IgM and IgG1 fractions were very little, if any, protection.     

Biological characterization of Campylobacter fetus lipopolysaccharides

Abstract Ten patients with chronic liver disease, seven healthy seropositive individuals with a remote history of rubella, and three patients with acute rubella were examined for serum levels of IgG subclasses and subclass antibodies against rubella virus structural proteins. One patient with AICAH had no detectable total or rubella specific IgG3 or IgG4. The liver disease patients were hypergammaglobulinemic and had greatly raised IgG1 levels. Patients with acute rubella lacked antibodies to the rubella virus E2 protein and showed no IgG4 antibody response. The liver disease patients showed a somewhat weaker IgG4 antibody response against the core (C) protein than healthy controls. However, differences are suggested within the subclasses in antibody reactivity against the individual rubella virus antigens. It is concluded that test systems that discriminate reactivities against individual antigens have to be used for characterization of viral antibody subclass profiles.     

Functional activity of different IgG subclass antibodies against type b capsular polysaccharide of Haemophilus influenzae

The biologic activity of different human IgG subclass antibodies directed against the Haemophilus influenzae type b (Hib) capsular polysaccharide (PRP) was compared by using an in vitro complement-mediated bactericidal assay and an in vivo passive protection assay in infant rats. An IgG pool was made by Sephacryl S-300 chromatography of sera from adults immunized with PRP vaccine. An IgG2 subclass fraction was prepared by column immunoabsorption of the IgG pool with anti-IgG1 monoclonal antibody. An IgG1 subclass fraction was eluted from the affinity matrix. IgG1, IgG2, IgG3, and IgG4 concentrations in the fractions were measured by solid-phase competitive radioimmunoassays, and anti-PRP antibody was measured by a modified Farr assay. Each fraction was greater than 90% pure IgG2 or IgG1, respectively. There were no significant differences in the minimal anti-PRP antibody concentrations required to kill 50% of Hib cells in vitro (IgG, 0.22; IgG1, 0.21; and IgG2, 0.42 microgram/ml). Similarly, equivalent amounts of anti-PRP antibody of the IgG1 or IgG2 fractions protected against bacteremia (IgG1, 0.12; IgG2, 0.24 microgram per rat). IgG absorbed to remove anti-PRP antibody was neither bactericidal nor protective. Thus IgG1 and IgG2 anti-PRP antibody have equivalent functional activities against Hib as determined by these biologic assays.     

Immunoglobulin G subclasses of anti-thyroid peroxidase autoantibodies in human autoimmune thyroid diseases

A distribution of immunoglobulin G (IgG) subclass of anti-thyroid peroxidase (TPO) autoantibodies was studied to know whether anti-TPO autoantibodies are closely implicated in the pathogenesis of human autoimmune thyroid diseases. As a result of analyzing 14 patients' sera, 7 with Graves' disease and 7 with Hashimoto's thyroiditis, anti-TPO autoantibodies were found to consist of mainly IgG1 subclass. Percentages of both IgG1 and IgG2 subclasses in IgG class of autoantibodies corresponded to those in the normal serum composition, whereas IgG3 subclass was scarcely contained in anti-TPO autoantibodies and IgG4 subclass markedly increased. It was thought that anti-TPO autoantibodies had a capability to lyse thyroid follicular cells by the mechanism of antibody-dependent complement-mediated cytolysis, because IgG1 and IgG2 subclasses of antibodies can fix complement and TPO locates in apical membrane surface of thyroid follicular cells. Comparing Graves' disease with Hashimoto's thyroiditis, mean percentages of both IgG1 and IgG2 subclasses of 2 groups were statistically different. Namely, sera of patients with Graves' disease had higher and lower mean percentages of IgG1 and IgG2 subclasses of autoantibodies, respectively, than those with Hashimoto's thyroiditis, though no plausible explanation for these differences can be offered at the present time.     

Antibody-mediated in vitro neutralization of human immunodeficiency virus type 1 abolishes infectivity for chimpanzees.

This study was undertaken to establish whether antibody directed against the human immunodeficiency virus type 1 (HIV-1) principal gp120 type-specific neutralization determinant can abolish the infectivity of HIV-1 in chimpanzees. Challenge inocula of the IIIb virus isolate were mixed in vitro with either immunoglobulin G (IgG) from an uninfected chimpanzee, nonneutralizing IgG from an HIV-seropositive human, a virus-neutralizing murine monoclonal antibody directed against the HIV-1 IIIb isolate, or virus-neutralizing IgG from a chimpanzee infected with the IIIb isolate. Both neutralizing antibodies were directed against the principal neutralization determinant of the challenge isolate. Establishment of infection following inoculation of each virus-antibody mixture into chimpanzees was assessed by virus-specific antibody development and by virus isolation. No protective effect was noted either with the control IgG or with the nonneutralizing anti-HIV IgG. By contrast, the polyclonal chimpanzee virus-neutralizing IgG prevented HIV-1 in vivo infection, while the neutralizing monoclonal antibody notably decreased the infectivity of the challenge virus. Hence, antibody to the gp120 principal neutralization determinant is able both to prevent HIV-1 infection in vitro and to inhibit infection in vivo.     

Measurement of serum levels of natalizumab, an immunoglobulin G4 therapeutic monoclonal antibody

Human immunoglobulin G4 (IgG4) is a poor trigger of effector functions and, therefore, is the preferred subclass for therapeutic monoclonal antibodies that merely aim to block their in vivo targets. An example is natalizumab, a recombinant IgG4 antibody directed against α4-integrin and used for treatment of multiple sclerosis. Efficient treatment requires that the pharmacokinetics of therapeutic monoclonal antibodies can be accurately monitored. For natalizumab, this requires special precautions due to recently reported structural peculiarities of human IgG4. Here we describe the development of an assay to determine serum levels of natalizumab. Compared with other IgG subclasses, human IgG4 possesses unique structural properties that influence its interactions in both in vivo and in vitro settings. Thus, IgG4 undergoes Fab arm exchange in vivo, resulting in effectively monovalent antibodies. Furthermore, IgG4 is able to bind to other human and nonhuman IgG via Fc interactions. We demonstrate how these features can interfere with measurement of specific IgG4 and describe how we addressed these issues, resulting in an assay that is not sensitive to Fab arm exchange by natalizumab or to IgG4 Fc interactions.     

Molecular and functional characterization of cynomolgus monkey IgG subclasses

Studies for vaccine and human therapeutic Ab development in cynomolgus monkeys (cynos) are influenced by immune responses, with Ab responses playing a significant role in efficacy and immunogenicity. Understanding the nature of cyno humoral immune responses and characterizing the predominant cyno IgG types produced and the Fc-FcγR interactions could provide insight into the immunomodulatory effects of vaccines. Anti-drug Ab responses against human IgG therapeutic candidates in cynos may affect efficacy and safety assessments because of the formation of immune complexes. There is, however, limited information on the structure and function of cyno IgG subclasses and how they compare with human IgG subclasses in Fc-dependent effector functions. To analyze the functional nature of cyno IgG subclasses, we cloned four cyno IgG C regions by using their sequence similarity to other primate IgGs. The four clones, cyno (cy)IGG1, cyIGG2, cyIGG3, cyIGG4, were then used to construct chimeric Abs. The sequence features of cyno IgG subclasses were compared with those of rhesus monkey and human IgG. Our data show that rhesus monkey and cyno IgG C regions are generally highly conserved, with differences in the hinge and hinge-proximal CH2 regions. Fc-dependent effector functions of cyno IgG subclasses were assessed in vitro with a variety of binding and functional assays. Our findings demonstrate distinctive functional properties of cyno IgG subclasses. It is notable that human IgG1 was less potent than cyno IgG1 in cyno FcγR binding and effector functions, with the differences emphasizing the need to carefully interpret preclinical data obtained with human IgG1 therapeutics.     

The in vitro resistance of IgG2 to proteolytic attack concurs with a comparative paucity of autoantibodies against peptide analogs of the IgG2 hinge

The mammalian antibody repertoire comprises immunoglobulin (Ig) molecules of multiple isotypes and subclasses with varying functional properties. Among the four subclasses of the human IgG isotype, we found that IgG2 exhibits a particular resistance to human and bacterial proteases that readily cleave the IgG1 hinge region in vitro. Autoantibodies (IgGs) that recognize points of proteolytic cleavage in the IgG1 hinge are widespread in the healthy human population, suggesting that IgG1 fragmentation and the generation of cryptic antigens for host immune surveillance commonly occur in vivo. We previously reported that autoantibodies to cleaved IgG1s can restore Fc-mediated effector functions that are lost following proteolytic cleavage of the hinge. In contrast, it was not possible to demonstrate an analogous cohort of autoantibodies to IgG2 hinge epitope analogs, and there appeared to be no functional component in human serum with the ability to reconstitute Fc effector functions to a cell-bound IgG2 fragment. Thus, the results indicate that among the IgG subclasses, human IgG2 is uniquely resistant to a number of known pathological proteases and that autoimmune recognition to potential cleavage points in the IgG2 hinge appears to be absent in human circulation.     

The subclass distribution of human IgG rheumatoid factor

The subclass distribution of IgG rheumatoid factor (RF) was determined by a sensitive ELISA assay in sera from patients with rheumatoid arthritis and from normal controls. In both instances, the most important subclasses were IgG1 and IgG4. The IgG4 RF was directed against the Fc region of IgG, and recognized human as well as rabbit IgG. Although human IgG4 myeloma proteins bound to rabbit IgG better than did myelomas of other IgG subclasses, the IgG4 RF activity in rheumatoid sera showed an additional specificity, because the fraction of IgG4 RF/total IgG4 for rheumatoid arthritis sera was far greater than for myelomas. This inference was supported by the observation that there was persistent, albeit diminished, IgG RF activity in pepsin-digested, RF-containing sera (but not myeloma proteins), indicating that a critical component of IgG4 RF activity was contained within the Fab region of the IgG4 molecule. The finding of large quantities of IgG4 RF was not due to a bias of the assay, because the preponderance of IgG4 did not extend to the subclass distribution of antibodies directed against other antigens. The demonstration of an important role for IgG4 as a RF is of special interest because of the relative inability of this subclass to fix complement or to bind to Fc receptors, and because of its potential role as a mediator of increased vascular permeability.     

The antibody response to the T1699 murine adenocarcinoma: antibody class and subclass heterogeneity detected in serum and in situ.

We have used indirect immunofluorescence to study antibody responses directed against membrane antigens expressed on in vitro and in vivo T1699 mammary adenocarcinoma cells. IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM antibodies were present in the serum of DBA/2 mice bearing T1699 tumors; IgG2a and IgG2b antibodies were readily detected on the cells in situ. Lesser amounts of the other classes and subclasses could be detected by indirect immunofluorescence measurements on in vivo tumor cells and with low pH eluates of in vivo cells tested on the in vitro line of T1699. The antigenic determinants on in situ tumor cells are not saturated with antibody as these cells demonstrated enhanced fluorescence of all immunoglobulin classes and subclasses when treated with autologous serum. Experiments with thymus-depleted mice indicated that immunoglobulin production was strongly dependent on thymus-derived cells for all immunoglobulin classes and subclasses except IgG2b. Our studies suggest that IgG2a may be active in the macrophage-mediated cytotoxic reaction and IgG2b in the immediate hypersensitivity reaction to T1699 cells. These results provide further evidence for an active role of tumor-specific antibody in the host defense to the T1699 adenocarcinoma in situ.