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α1,6-Fucosyltransferase (Fut8) is the enzyme responsible for catalyzing core fucosylation. Exogenous L-fucose upregulates fucosylation levels through the GDP-fucose salvage pathway. This study investigated the relationship between core fucosylation and immunoglobulin G (IgG) amounts in serum utilizing WT (Fut8 +/+ ), Fut8 heterozygous knockout (Fut8 +/- ), and Fut8 knockout (Fut8 -/- ) mice. The IgG levels in serum were lower in Fut8 +/- and Fut8 -/- mice compared with Fut8 +/+ mice. Exogenous L-fucose increased IgG levels in Fut8 +/- mice, while the ratios of core fucosylated IgG versus total IgG showed no significant difference among Fut8 +/+ , Fut8 +/- , and Fut8 +/- mice treated with L-fucose. These ratios were determined by Western blot, lectin blot, and mass spectrometry analysis. Real-time PCR results demonstrated that mRNA levels of IgG Fc and neonatal Fc receptor, responsible for protecting IgG turnover, were similar among Fut8 +/+ , Fut8 +/- , and Fut8 +/- mice treated with L-fucose. In contrast, the expression levels of Fc-gamma receptor Ⅳ (FcγRⅣ), mainly expressed on macrophages and neutrophils, were increased in Fut8 +/- mice compared to Fut8 +/+ mice. The effect was reversed by administrating L-fucose, suggesting that core fucosylation primarily regulates the IgG levels through the Fc-FcγRⅣ degradation pathway. Consistently, IgG internalization and transcytosis were suppressed in FcγRⅣ-knockout cells while enhanced in Fut8-knockout cells. Furthermore, we assessed the expression levels of specific antibodies against ovalbumin and found they were downregulated in Fut8 +/- mice, with potential recovery observed with L-fucose administration. These findings confirm that core fucosylation plays a vital role in regulating IgG levels in serum, which may provide insights into a novel mechanism in adaptive immune regulation., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Antibodies can mediate immune recruitment or clearance of immune complexes through the interaction of their Fc domain with cellular Fc receptors. Clustering of antibodies is a key step in generating sufficient avidity for efficacious receptor recognition. However, Fc receptors may be saturated with prevailing, endogenous serum immunoglobulin and this raises the threshold by which cellular receptors can be productively engaged. Here, we review the factors controlling serum IgG levels in both healthy and disease states, and discuss how the presence of endogenous IgG is encoded into the functional activation thresholds for low- and high-affinity Fc receptors. We discuss the circumstances where antibody engineering can help overcome these physiological limitations of therapeutic antibodies. Finally, we discuss how the pharmacological control of Fc receptor saturation by endogenous IgG is emerging as a feasible mechanism for the enhancement of antibody therapeutics., Competing Interests: Declaration of Competing Interest S.A.B. has acted as a consultant for a number of biotechs and receives institutional support for grants and patents from BioInvent. M.C. has acted as a consultant for a number of biotechs and legal firms and is a named inventor on a patent application describing “Combined therapeutic use of antibodies and endoglycosidases” (WO2013110946A1) which was acquired by Hansa Biopharma., (Copyright © 2023. Published by Elsevier B.V.)

The dermis is the portal of entry for most vector-transmitted pathogens, making the host's immune response at this site critical in mitigating the magnitude of infection. For malaria, antibody-mediated neutralization of Plasmodium parasites in the dermis was recently demonstrated. However, surprisingly little is known about the mechanisms that govern antibody transport into the skin. Since the neonatal Fc receptor (FcRn) has been shown to transcytose IgG into various tissues, we sought to understand its contribution to IgG transport into the skin and antibody-mediated inhibition of Plasmodium parasites following mosquito bite inoculation. Using confocal imaging, we show that the transport of an anti-Langerin mAb into the skin occurs but is only partially reduced in mice lacking FcRn. To understand the relevance of FcRn in the context of malaria infection, we use the rodent parasite Plasmodium berghei and show that passively-administered anti-malarial antibody in FcRn deficient mice, does not reduce parasite burden to the same extent as previously observed in wildtype mice. Overall, our data suggest that FcRn plays a role in the transport of IgG into the skin but is not the major driver of IgG transport into this tissue. These findings have implications for the rational design of antibody-based therapeutics for malaria as well as other vector-transmitted pathogens., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Nasir, Sinnis. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Although interactions of small molecular drugs with serum proteins have been widely studied from pharmacokinetic and pharmacodynamic perspectives, there have been few reports on the effects of serum components on therapeutic antibody functions. This study reports the effect of abundant serum proteins on antibody-dependent cellular cytotoxicity (ADCC) mediated by rituximab and Fcγ receptor III (FcγRIII). Human serum albumin (HSA) and the Fab fragment from the pooled serum polyclonal IgG were found to compromise ADCC as non-competitive inhibitors. Our nuclear magnetic resonance data provided direct evidence for the interactions of HSA with both the Fab and Fc regions of rituximab and also with the extracellular region of FcγRIII (sFcγRIII). The degree of involvement in the interaction decreased in the order of rituximab-Fab > rituximab-Fc > sFcγRIII, suggesting preferential binding of HSA to net positively charged proteins. Although much less pronounced than the effect of HSA, polyclonal IgG-Fab specifically interacted with rituximab-Fc. The NMR data also showed that the serum protein interactions cover the Fc surface extensively, suggesting that they can act as pan-inhibitors against various Fc receptor-mediated functions and pharmacokinetics. Our findings highlight the importance of considering serum-protein interactions in the design and application of antibody-based drugs with increased efficacy and safety., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yanaka, Yogo, Yagi, Onitsuka, Wakaizumi, Yamaguchi, Uchiyama and Kato.)

In the last decades, antibody-based tumor therapy has fundamentally improved the efficacy of treatment for patients with cancer. Currently, almost all tumor antigen-targeting antibodies approved for clinical application are of IgG1 Fc isotype. Similarly, the mouse homolog mIgG2a is the most commonly used in tumor mouse models. However, in mice, the efficacy of antibody-based tumor therapy is largely restricted to a prophylactic application. Direct isotype comparison studies in mice in a therapeutic setting are scarce. In this study, we assessed the efficacy of mouse tumor-targeting antibodies of different isotypes in a therapeutic setting using a highly systematic approach. To this end, we engineered and expressed antibodies of the same specificity but different isotypes, targeting the artificial tumor antigen CD90.1/Thy1.1 expressed by B16 melanoma cells. Our experiments revealed that in a therapeutic setting mIgG2a was superior to both mIgE and mIgG1 in controlling tumor growth. Furthermore, the observed mIgG2a antitumor effect was entirely Fc mediated as the protection was lost when an Fc-silenced mIgG2a isotype (LALA-PG mutations) was used. These data confirm mIgG2a superiority in a therapeutic tumor model., Significance: Direct comparisons of different antibody isotypes of the same specificity in cancer settings are still scarce. Here, it is shown that mIgG2a has a greater effect compared with mIgG1 and mIgE in controlling tumor growth in a therapeutic setting., Competing Interests: N. Vukovic reports grants and personal fees from European Union's Horizon 2020 research and innovation programme during the conduct of the study. M. Waterfall reports there are no relationships etc. that M. Waterfall believes are a conflict of interest. However, your form will not allow the selection of the NO check box. D.M. Zaiss reports grants and personal fees from European Union's Horizon 2020 during the conduct of the study. No disclosures were reported by the other authors., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Serum albumin (SA), the most abundant soluble protein in the body, maintains plasma oncotic pressure and regulates the distribution of vascular fluid and has a range of other important functions. The goals of this review are to expand clinical knowledge regarding the functions of SA, elucidate effects of dysregulated SA concentration, and discuss the clinical relevance of hypoalbuminemia resulting from various diseases. We discuss potential repercussions of SA dysregulation on cholesterol levels, liver function, and other processes that rely on its homeostasis, as decreased SA concentration has been shown to be associated with increased risk for cardiovascular disease, hyperlipidemia, and mortality. We describe the anti-inflammatory and antioxidant properties of SA, as well as its ability to bind and transport a plethora of endogenous and exogenous molecules. SA is the primary serum protein involved in binding and transport of drugs and as such has the potential to affect, or be affected by, certain medications. Of current relevance are antibody-based inhibitors of the neonatal Fc receptor (FcRn), several of which are under clinical development to treat immunoglobulin G (IgG)-mediated autoimmune disorders; some have been shown to decrease SA concentration. FcRn acts as a homeostatic regulator of SA by rescuing it, as well as IgG, from intracellular degradation via a common cellular recycling mechanism. Greater clinical understanding of the multifunctional nature of SA and the potential clinical impact of decreased SA are needed; in particular, the potential for certain treatments to reduce SA concentration, which may affect efficacy and toxicity of medications and disease progression., Competing Interests: DG, EL, and JVS are employees of argenx, the manufacturer of efgartigimod (FDA approved in gMG and under investigation in primary immune thrombocytopenia, pemphigus vulgaris and foliaceus, chronic inflammatory demyelinating polyneuropathy, bullous pemphigoid, and idiopathic inflammatory myopathy [myositis]). JTA has received funding from Syntimmune and argenx as part of fee-for-service agreements. ED is a postdoctoral research fellow of the Research Foundation – Flanders (grant number 12X9420N). JEK has participated on the Medical Advisory Committee of Sanofi Genzyme and Plasma Advisory Committee of Haemonetics. DR is the founder of Staten Biotechnology. NS is a member of the scientific advisory board and a speaker for argenx. IS, is supported by the Clinical Research Fund of the University Hospitals Leuven. ESW may receive royalties from patents owned by the UK Medical Research Council, UT Southwestern Medical Center, and Texas A&M University, and has financial interests in argenx, Astero BioPharma LLC, Astero Erado Inc., and Astero Technologies LLC. GV is a paid consultant for argenx. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The development (medical writing and editorial support) of this manuscript received funding from argenx. Authors who are not affiliated with argenx were not paid for their contributions to this work. The funder was involved in conception of design, interpretation of data, writing of this article, and decision to submit it for publication., (Copyright © 2022 Ward, Gelinas, Dreesen, Van Santbergen, Andersen, Silvestri, Kiss, Sleep, Rader, Kastelein, Louagie, Vidarsson and Spriet.)

Humans have four IgG antibody subclasses that selectively or differentially engage immune effector molecules to protect against infections. Although IgG1 has been studied in detail and is the subclass of most approved antibody therapeutics, increasing evidence indicates that IgG3 is associated with enhanced protection against pathogens. Here, we report that IgG3 has superior capacity to mediate intracellular antiviral immunity compared with the other subclasses due to its uniquely extended and flexible hinge region, which facilitates improved recruitment of the cytosolic Fc receptor TRIM21, independently of Fc binding affinity. TRIM21 may also synergize with complement C1/C4-mediated lysosomal degradation via capsid inactivation. We demonstrate that this process is potentiated by IgG3 in a hinge-dependent manner. Our findings reveal differences in how the four IgG subclasses mediate intracellular immunity, knowledge that may guide IgG subclass selection and engineering of antiviral antibodies for prophylaxis and therapy.

Advances in antibody engineering have enabled the construction of novel molecular formats in diverse shapes and sizes, providing new opportunities for biologic therapies and expanding the need to understand how various structural aspects affect their distribution properties. To assess the effect of antibody size on systemic pharmacokinetics (PK) and tissue distribution with or without neonatal Fc receptor (FcRn) binding, we evaluated a series of non-mouse-binding anti-glycoprotein D monoclonal antibody formats, including IgG [~150 kDa], one-armed IgG [~100 kDa], IgG-HAHQ (attenuated FcRn binding) [~150 kDa], F(ab') 2 [~100 kDa], and F(ab) [~50 kDa]. Tissue-specific concentration-time profiles were corrected for blood content based on vascular volumes and normalized based on interstitial volumes to allow estimation of interstitial concentrations and interstitial:serum concentration ratios. Blood correction demonstrated that the contribution of circulating antibody on total uptake was greatest at early time points and for highly vascularized tissues. Tissue interstitial PK largely mirrored serum exposure profiles. Similar interstitial:serum ratios were obtained for the two FcRn-binding molecules, IgG and one-armed IgG, which reached pseudo-steady-state kinetics in most tissues. For non-FcRn-binding molecules, interstitial:serum ratios changed over time, suggesting that these molecules did not reach steady-state kinetics during the study. Furthermore, concentration-time profiles of both intact and catabolized molecule were measured by a dual tracer approach, enabling quantification of tissue catabolism and demonstrating that catabolism levels were highest for IgG-HAHQ. Overall, these data sets provide insight into factors affecting preclinical distribution and may be useful in estimating interstitial concentrations and/or catabolism in human tissues.

Fc-mediated effector functions are important for the clearance of pathologic cells by therapeutic IgG antibodies through two mechanisms: via the activation of the classical complement pathway and through the binding to Fcγ receptors (FcγRs) which mediate clearance of targeted cells by antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) by effector cells such as macrophages, NK cells, and other leukocytes subsets. Complement activation results in direct cell killing through the formation of the membrane attack complex (MAC, complement-dependent cytotoxicity or CDC) and in the deposition of complement opsonins on pathogen surfaces. The latter are recognized by complement receptors on effector cells in turn triggering complement-dependent cell cytotoxicity and phagocytosis (CDCC and CDCP, respectively). Little is known about the role of CDCC and CDCP on therapeutic antibody function because on the one hand, IgG isotype antibodies bind to both FcγR and C1q to activate the complement pathway, and on the other, immune cells express complement receptor as well as FcγRs. We engineered IgG1 Fc domains that bind with high affinity to C1q but have very little or no binding to FcγR. To this end, we employed display of IgG in E. coli (which lack protein glycosylation machinery) for the screening of very large libraries (>2 × 10 9 ) of randomly mutated human Fc domains to isolate Fc variants that bind to C1q. Herein we introduce and describe the method., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

The MHC class I-related receptor FcRn serves multiple roles ranging from the regulation of levels of IgG isotype antibodies and albumin throughout the body to the delivery of antigen into antigen loading compartments in specialized antigen-presenting cells. In parallel with studies directed towards understanding FcRn at the molecular and cellular levels, there has been an enormous expansion in the development of engineering strategies involving FcRn to modulate the dynamic behavior of antibodies, antigens, and albumin. In this review article, we focus on a discussion of FcRn-targeted approaches that have resulted in the production of novel antibody-based platforms with considerable potential for use in the clinic., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Anti-neutrophil cytoplasmic antibody (ANCA) vasculitis is characterized by the presence of autoantibodies to myeloperoxidase and proteinase-3, which bind monocytes in addition to neutrophils. While a pathological effect on neutrophils is acknowledged, the impact of ANCA on monocyte function is less well understood. Using IgG from patients we investigated the effect of these autoantibodies on monocytes and found that anti-myeloperoxidase antibodies (MPO-ANCA) reduced both IL-10 and IL-6 secretion in response to LPS. This reduction in IL-10 and IL-6 depended on Fc receptors and enzymatic myeloperoxidase and was accompanied by a significant reduction in TLR-driven signaling pathways. Aligning with changes in TLR signals, oxidized phospholipids, which function as TLR4 antagonists, were increased in monocytes in the presence of MPO-ANCA. We further observed that MPO-ANCA increased monocyte survival and differentiation to macrophages by stimulating CSF-1 production. However, this was independent of myeloperoxidase enzymatic activity and TLR signaling. Macrophages differentiated in the presence of MPO-ANCA secreted more TGF-β and further promoted the development of IL-10- and TGF-β-secreting CD4 + T cells. Thus, MPO-ANCA may promote inflammation by reducing the secretion of antiinflammatory IL-10 from monocytes, and MPO-ANCA can alter the development of macrophages and T cells to potentially promote fibrosis., Competing Interests: The authors have declared that no conflict of interest exists.

Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine-induced antibodies and therapeutic antibodies will enable a better understanding of their capacity to prevent and/or control HIV-1 infection in vivo., Competing Interests: The authors have declared that no competing interests exist.

Nearly 350 IgG-based therapeutics are approved for clinical use or are under development for many diseases lacking adequate treatment options. These include molecularly engineered biologicals comprising the IgG Fc-domain fused to various effector molecules (so-called Fc-fusion proteins) that confer the advantages of IgG, including binding to the neonatal Fc receptor (FcRn) to facilitate in vivo stability, and the therapeutic benefit of the specific effector functions. Advances in IgG structure-function relationships and an understanding of FcRn biology have provided therapeutic opportunities for previously unapproachable diseases. This article discusses approved Fc-fusion therapeutics, novel Fc-fusion proteins and FcRn-dependent delivery approaches in development, and how engineering of the FcRn-Fc interaction can generate longer-lasting and more effective therapeutics.

Fc receptor-like A (FCRLA) is an unusual member of the extended Fc receptor family. FCRLA has homology to receptors for the Fc portion of Ig (FCR) and to other FCRL proteins. However, unlike these other family representatives, which are typically transmembrane receptors with extracellular ligand-binding domains, FCRLA has no predicted transmembrane domain or N-linked glycosylation sites and is an intracellular protein. We show by confocal microscopy and biochemical assays that FCRLA is a soluble resident endoplasmic reticulum (ER) protein, but it does not possess the amino acid sequence KDEL as an ER retention motif in its C-terminus. Using a series of deletion mutants, we found that its ER retention is most likely mediated by the amino terminal partial Ig-like domain. We have identified ER-localized Ig as the FCRLA ligand. FCRLA is unique among the large family of Fc receptors, in that it is capable of associating with multiple Ig isotypes, IgM, IgG and IgA. Among hemopoietic cells, FCRLA expression is restricted to the B lineage and is most abundant in germinal center B lymphocytes. The studies reported here demonstrate that FCRLA is more broadly expressed among human B lineage cells than originally reported; it is found at significant levels in resting blood B cells and at varying levels in all B-cell subsets in tonsil.

Gram-positive bacterial pathogens express immunoglobulin (Ig) binding proteins that perturb Fc-dependent functions such as the interaction with complement or phagocytic Fc-receptors. The possession of such molecules by gram-negative bacteria, including Escherichia coli (E. coli), has also been documented. In many such studies, the detection of Ig binding has relied on the use of polyclonal antibodies as detecting reagents. These are not ideal since such preparations may be contaminated with E. coli specific IgG, allowing for the potential misinterpretation of specific F(ab')(2) binding as non-specific Fc mediated binding. Here we use mono-specific recombinant antibodies to develop a novel assay for Ig binding non reliant on traditional polyclonal antibodies, allowing us to demonstrate the unequivocal absence of Fc binding proteins from E. coli.

Although immobilization of antigen-specific immunoglobulins onto matrix-assisted laser desorption/ionization (MALDI) targets allows the specific detection and enrichment of an antigen from complex biological fluids, the process of antibody immobilization is not optimal. The principal reason is that the antibody can bind to the template in various orientations, many of which block antigen recognition. An affinity capture MALDI mass spectrometry methodology was developed by covalently immobilizing an Fc receptor (recombinant protein G) onto MALDI gold targets for the purpose of orientating an immunoglobulin G, with the Fab domains pointing away from the target surface. The pregnancy and cancer marker, human chorionic gonadotropin beta core fragment (hCGbetacf), was our chosen test substance. To optimize the methodology, different surface densities of protein G and immunoglobulin were achieved by employing varying concentrations for immobilization. Captured amounts of hCGbetacf were compared using an external standard (cytochrome c). Orientation of immunoglobulin resulted in an approximately 3-fold increase in MALDI signal compared to using randomly immobilized antibody. Higher antibody concentrations resulted in diminished MALDI signals, which were explained by steric hindrance. Purification and enrichment of hCGbetacf was achieved from a test solution containing contaminant peptides and proteins using oriented immunoglobulins on-target.

In order to study placental transfer of IgG subclasses, paired blood samples were collected from mothers and umbilical cord of preterm (N = 69) and full-term (N = 68) newborns. The full-term group was further divided into 3 subgroups: appropriate for gestational age (AGA, N = 43), large for gestational age (LGA, N = 13) and small for gestational age (SGA, N = 12), according to birth weight. IgG subclasses (IgG1, IgG2, IgG3 and IgG4) were measured by the single radial immunodiffusion technique using monoclonal antibodies. IgG1 and IgG3 newborn subclass concentrations (10.17 and 0.57 g/l, respectively) increased with increasing gestational age and reached maternal levels (IgG1 = 8.86; IgG3 = 0.67 g/l) during the 37th week of pregnancy. Low levels of these subclasses were found in premature newborns. IgG2 from newborns were always lower than maternal levels (P < 0.05). LGA and SGA newborns had equivalent levels of IgG1 and IgG2 compared with AGA. SGA newborns had higher levels of IgG3 and lower levels of IgG4 than LGA and AGA newborns.

Case of rheumatoid arthritis (RA) with interstitial lung disease (ILD) have been reported to show increased titers of rheumatoid factor (RF) in serum; however the pathogenic role of this substance in the lung is still obscure. The aims of this study were to estimate IgGRF in the bronchoalveolar lavage fluid (BALF) in RA and to investigate its possible roles in the ILD of RA. Two step bronchoalveolar lavage in 20 RA patients (9 males, 11 females) was performed in conjunction with high resolution chest CT. IgGRF was measured by enzyme-linked immunosorbent assay and its molecular size in BALF was evaluated by high performance liquid chromatography. IgGRF bound to BALF cells was eluted by sonication of the cells. RA patients with ILD had significantly elevated levels of IgGRF in serum as well as in BALF. The IgGRF in BALF was monomeric in 2 of 3 RA patients with ILD. The activity of IgGRF bound to BALF cells was significantly higher in RA with ILD than in control patients. These findings suggest that monomeric IgGRF in the lungs of RA patients could bind to the cells with Fc receptors.

IgM antibodies have previously been reported to either inhibit or induce antibody-dependent lymphocyte cytotoxicity (ADCC). Here we show that human lymphocytes lyse bovine erythrocytes (Eb) in the presence of either IgM of IgG anti-Eb from rabbits. Seven out of 20 IgM preparations (Sephadex G-200) were ADCC-active. IgG-dependent ADCC was inhibited by human IgG but not by IgM. In contrast, IgM ADCC was inhibited by both IgG and IgM. The effector cells in IgM ADCC were a subpopulation of lymphocytes with distinct Fc receptors for both IgG and IgM. Most of them also had sheep erythrocyte receptors. Extensive purification of the ADCC-active IgM antibody preparations indicated that very small amounts of contaminating IgG anti-Eb were responsible for ADCC induction. When purified and ADCC-inactive IgM antibodies were mixed with suboptimal concentrations of IgG antibodies, a strong enhancement of ADCC was found. To achieve enhancement, the two antibody isotypes had to be present on the surface of the same target cells, and the IgM effect was not due to the release of soluble ADCC-enhancing factors. Thus, in this system, IgM antibodies are not capable of inducing ADCC on their own. However, they enhance ADCC by improving the contactual interaction between target cells and a special subset of effector cells.

The presence of IgG Fc receptors in the tissue sections of aorta and aortic valves of man, ox and pig was demonstrated by means of hemadsorption of human and rabbit IgG antibody sensitized erythrocytes to these sections. C3b receptors in the tissue sections of aortic valves of these three mammals were also demonstrated by using sheep erythrocytes sensitized with rabbit IgM antibody which bound murine complement. 8 of 13 systemic lupus erythematosus sera and 2 of 19 other collagen disease sera were shown to be able to inhibit the adsorption of rabbit IgG antibody sensitized erythrocytes to the human aortic sections upon preincubation of the tissues with the pathologic sera. The amount of immune complexes in the 'inhibition-positive' sera was significantly higher than that in the negative sera.

By two independent techniques for separating human opsonic IgG for group A type 6 streptococci into fast- and slow-migrating fractions, it was found that the opsonic activity was localized within the basic charge population. This charge dependence was found to be a characteristic of the IgG isolated from three individuals. When the fast- and slow-migrating IgG fractions were tested for their ability to bind to purified M6 protein, antibodies in both opsonic and nonopsonic populations exhibited binding activity, with the majority being located within the opsonic IgG in two of the three individuals; the third displayed greater binding in the nonopsonic population. The functional difference observed in the antibody populations to this M antigen may be a reflection of the net charge within the area of the antibody binding site, which suggests that the opsonic antibodies need to bind to acidic residues along the outer surface of the fibrillar M protein molecule. F(ab')2 fragments prepared from both human and rabbit type 6 opsonic IgG were still able to bind to the M6 molecule but were unable to mediate opsonization of type 6 streptococci. However, the F(ab')2 fragments had the capacity to enhance or amplify the opsonic activity of low concentrations of opsonic IgG molecules. The results suggest that the M protein molecule may function as an active inhibitor of phagocytosis and that F(ab')2 fragments from opsonic IgG have the capacity to neutralize the "active" determinants on the molecule, thus allowing lower concentrations of IgG with functional Fc receptors to mediate phagocytosis.

Enumeration of a subpopulation of T cells with receptors for the Fc portion of IgG (T gamma) in the peripheral blood of 14 normal subjects and 43 patients with leprosy was undertaken. Tuberculoid leprosy patients showed normal levels of T gamma cells. In contrast, bacillary positive patients with lepromatous leprosy revealed a significant reduction of circulating T gamma cells (p less than 0.001).

Since the U937 cell line expresses many characteristics of normal human monocytes and macrophages, we studied in detail its receptors for IgG by measuring direct binding and inhibition of binding of 16 radioiodinated human myeloma proteins representative of the 4 subclasses. As with normal monocytes, IgG1 and IgG3 bound most efficiently (17.3 and 15.7% bound), IgG4 less readily (7.1% bound), and IgG2 least readily (0.6% bound). Scatchard plots of IgG1 binding showed approximately 18,000 binding sites/cell with Ka approximately 10(8) liter/mol. IgG1 binding was inhibited equally well by IgG1 and IgG3 (50% inhibition with 0.26 +/- 0.03 and 0.26 +/- 0.09 microgram, respectively). IgG4 inhibited less readily (0.68 +/- 0.12 microgram). Three IgG2 proteins were not inhibitory (115 +/- 59) but one IgG2 myeloma inhibited well (0.89 +/- 0.47). IgG Fc fragments inhibited IgG1 binding 1000-fold more efficiently than Fab fragments, IgM, and IgA. Reciprocal inhibition experiments gave no indication of multiple receptor sites of differing specificities. In situ, the IgG1 receptor was resistant to proteases. The data suggest that the U937 Fc receptor may be a useful model of human macrophage structure and function.

The interaction of IgG with Fc gamma receptors on human PMN was studied by investigating the capacity of IgG and some of its fragments to mediate and inhibit, respectively, binding of immune complexes to these receptors and the subsequent activation of the cells. Fragments included Fab/Fc which is monovalent but contains the complete Fc region, Facb (divalent, C gamma 3 domains removed), Fc, pFc' (a C gamma 3 dimer) and a peptide corresponding to the greater part of one C gamma 2 domain (amino acids 278 through 333) including the carbohydrate side chains. F(ab')2 and Fab served as controls. The results indicate that human IgG interacts with human PMN Fc receptors predominantly through its C gamma 2 domains, while binding of rabbit IgG involves additional sites in C gamma 3. The C gamma 2 binding site on human IgG appears to be located between those for C1 and protein A and to contain lysine residues. For its correct exposure the C gamma 2 domains must be kept in the native conformation, in which both the hinge disulfide bridges and the carbohydrate moieties are involved. A role of the sugars in the actual binding, on the other hand, can be excluded.

One unambiguous message that comes from the data obtained relates to the central role played by the hinge region, and its constituent disulfide bonds, in the functioning of the IgG molecule. Segmental flexibility allows variation in the distance between the antibody combining sites, located at the distal ends of the Fab regions, and serves the important function of allowing bivalent attachment of antibodies to antigenic determinants even if the latter form a fixed array, for example, on a cell surface (monogomous bivalency). Too much flexibility, however, seen as a consequence of cleaving the hinge-region disulfides appears to be inimicable to the expression of effector functions. The hinge region and its disulfides control the degree of flexibility, thus allowing the IgG molecule to perform its varied functions.

Abnormally low proportions of total T lymphocytes were found in peripheral blood lymphocytes (PBL) of patients with multiple sclerosis (MS) accompanied by high proportions of T lymphocytes with Fc receptors (FcR) for IgG (T gamma cells). Seventy-five percent of the MS patients had greater than 40% T gamma in the T lymphocyte-enriched population. In 6 out of 16 cases, T mu were also elevated. This elevation, however, did not seem to correlated with disease stage. By mixed rosetting and direct observation with an indirect immunofluorescence test, 30 to 50% of the T gamma cells in MS PBL were also shown to bear FcR for IgM. This population bearing FcR for IgG and IgM, designated T gamma mu, was usually found to be 10% or less of the T gamma population in normal controls. The significance of this T gamma mu population for the MS disease process requires further evaluation.

The ultrastructural and cytochemical features of human peripheral blood TG cells (T cells with receptors IgG) and of the cells of the so-called third population (non-T, non-B cells with high avidity receptors for IgG) have been investigated and compared. Both TG and third-population cells (TPC) contained acid hydrolases with a paranuclear localization of alpha-naphthyl acid esterase, beta-glucuronidase or acid phosphatase. At the electron microscopy level, TG and TPC were indistinguishable and displayed rough cell surface, indented nuclei, abundant cytoplasm with predominance of the smooth over the rough membranes and peroxidase-negative granules. A large proportion of cells of the TPC could form rosettes with sheep erythrocytes after treatment with neuraminidase. The observed close similarities between TG and TPC may suggest that both cell types belong to a special subset of T cells. However, the alternative hypothesis that both TG and TPC are part of a subset unrelated to T cells, such as a new non-T, non-B cell population, or even of the monocytic-macrophage lineage, is also discussed.

Binding studies of human IgG proteins to murine P388D1 cells indicated that they bind to an apparently homogeneous Fc receptor population. The association constant was 0.89 x 10(6)M-1 at 22 degrees C and was comparable to the binding affinities of homologous murine IgG2a and IgG2b. The number of receptor sites was found to be approximately 6 x 10(5)/cell. Fc gamma 1 and Fc gamma 3 fragments bound with an affinity comparable to that of the parent proteins. The P388D1 receptors could discriminate between the human IgG subclasses; the relative cytophilic activity was IgG3 greater than IgG1 greater than IgG4 and IgG2 was devoid of binding activity. Fragments corresponding to the C gamma 2 and C gamma 3 domains of human IgG1 were both unable to bind to the P388D1 receptors either alone or in equimolar combination. This suggests that the cytophilic site may be formed cooperatively by interaction between the two domains. The integrity of the hinge region appeared to be essential for full expression of cytophilic activity since reduction of the hinge-region disulfides in both human IgG1 and its Fc fragment markedly decreased their binding affinity. In addition, a mutant IgG1 molecule lacking the hinge region was significantly less cytophilic than its normal counterpart.

A human mononuclear cell population, enriched with monocytes by adhering them to microexudate on Petri dishes previously used for fibroblast cultures was used in a homologous human system to form EA rosettes. IgG1, Fc, and subfragments of Fc representing the C gamma 2 and C gamma 3 domains were tested for their ability to inhibit rosette formation. Although Fc and IgG were equally effective in inhibiting rosette formation over the concentration range 10(-6) to 10(-5) M, all subfragments were inactive at these concentrations. At 10-fold higher concentrations the C gamma 2 fragment was still inactive; however, both the C gamma 3 fragments, pFc' and at C gamma 3, did show significant activity at this higher level. Reduction and alkylation diminished the inhibitory capacity of IgG 10-fold but had a lesser effect on Fc. In a parallel series of experiments the ability of IgG and Fc to inhibit granulocyte rosettes was found to be markedly diminished by reduction and alkylation in both cases. These experiments reveal differences between Fc receptors in different cells, confirm a role for the C gamma 3 homology region in monocyte Fc receptor recognition, but do suggest a requirement, either direct or indirect, for the C gamma 2 domain.

The number of free Fc receptors (FcR) per cell and the association constant (Kass) for the binding of monomeric IgG were determined for monocyte-enriched peripheral blood mononuclear cells, isolated from 16 patients with active classical rheumatoid arthritis (RA) and from 15 normal healthy donors. The assay system was based on binding under equilibrium conditions of 125I-labelled monomeric rabbit IgG to monocytes purified from peripheral blood on a continuous gradient of Percoll. Monocytes from 14 untreated RA patients (6 seropositive, 8 seronegative) expressed on the average 4.8 +/- 1.3 x 10(4) FcR/cell. This number was significantly higher (P less than 0.001) than that found in the control group (34. +/- 0.7 x 10(4) FcR/cell). There was also a significant difference between the mean Kass of the RA group and the control group--2.1 +/- 0.7 x 10(8) l/mol and 2.6 +/- 1.0 x 10(8) l/mol, respectively (0.05 greater than P greater than 0.01). Two seropositive RA patients receiving systemic treatment with penicillamine expressed the same number of FcR/cell as the mean of the control group (3.6 x 10(4)). Levels of circulating immune complexes (CIC) and the complement-factor C3 split product C3d were also measured. No correlation was found between the number of FcR/cell and the concentration of C3d, but there was a weak correlation between the number of FcR/cell and the level of CIC.

Previous studies have indicated that adult schistosomes have an affinity for both IgG and C; however, thorough investigations on the possible presence of receptors for these serum proteins on the adult schistosome tegument have not been attempted. The present study presents evidence for both IgG-Fc and C3 receptors on adult schistosomes. An indirect assay utilizing fluoresceinated Staphylococcus aureus was developed to detect antigen-antibody (Ag-Ab) and antigen-antibody-C (Ag-Ab-C) binding to adult parasites. Detection of Ag-Ab and Ag-Ab-C complex binding was dependent on the presence of a metabolic inhibitor in the incubation mixture presumably to prevent shedding of bound complexes. Ag-Ab complex binding required the presence of an intact Fc on the IgG of the complex and could be partially inhibited by preincubation of parasites in heat-aggregated IgG or an isolated Fc preparation. Binding of Ag-Ab-C complexes was dependent on active C3 in the C source but not on an intact Fc on the IgG of the complex. Ag-Ab-C binding could be blocked by prior incubation of parasites in heat-aggregated IgG-C but not by C alone. Binding of host serum proteins to IgG-Fc and C3 receptors on adult schistosomes may aid parasite survival by helping to prevent immune detection.

Intact syncytiotrophoblast microvillous plasma membrane (StMPM) vesicle preparations isolated from human term placentae express surface Fc gamma receptor activity and will bind to Sepharose-immobilised human IgG. This IgG-StMPM Fc gamma receptor interaction has been shown to be stable during de-membranation using the anionic detergent 1% sodium deoxycholate (NaDOC) and to be preferentially contained in an NaDOC-resistant fraction of StMPM vesicles. Following NaDOC treatment of StMPM adsorbed on to Sepharose-IgG, subsequent desorption with the chaotrope 0.5 M NH4SCN disrupted the IgG-Fc gamma receptor interaction. The NH4SCN desorption product, however, contained several molecular components including IgG, transferrin and actin. These results indicate that StMPM surface Fc gamma receptors may interact with proteins of the microfilamentous core of StMPM vesicles and also possibly be topographically associated with some other cell surface components.

Normal human lymphocytes lyse a variety of erythrocytes when effector cells and target cells are incubated on immobilized, normal human IgG aggregates (10). Lymphocyte-mediated cytolysis induced by immobilized aggregate requires an overnight incubation with adherent mononuclear cells. This study was carried out to determine whether preincubation with adherent cells effects the activation of a cytolytic cell or inactivation of a suppressor cell. It was determined that a suppressor cell function is inactivated, and the suppressor cells were characterized as T gamma and T mu lymphocytes.

Receptors for IgG and IgE on rat basophilic leukemia cells (the 2H3 subline of RBL-IV) were detected by their ability bind IgE and IgG in fluorescence or radioactive assays. The 2H3 cells have approximately two-thirds as many receptors for IgG as for IgE. The IgE receptors bind monomeric IgE with high affinity and IgE binding is uninhibited by high concentrations of monomeric or oligomeric IgG. In contrast, the IgG receptors bind both (rabbit and rat) IgG and (rat) IgE. The affinity of IgG receptors for monomeric IgG and IgE is much lower than that of the IgE receptor for its ligand; binding of radiolabeled IgG or IgE to IgG receptors can be detected only with cross-linked oligomers. Although both receptors are sensitive to tryptic digestion than IgE receptors. Dual laser flow microfluorometric studies revealed that the numbers of IgG and IgE receptors per cell varied considerably among 2H3 cells and that the distributions of the 2 types of receptors were independent of each other. 2H3 cells released histamine through an IgE-mediated system, but cross-linked IgG, even in saturating amounts, did not elicit histamine release, nor have any effect upon IgE-induced release. Moreover, 2H3 cells would not mediate ADCC of IgG-coated chicken red blood cells.

The results of previous studies in our laboratory have shown that mice bearing plasmacytomas and hybridomas that secrete IgA or IgE are accompanied by increased frequencies of Lyt-1-2+ T lymphocytes bearing Fc receptors (FcR) for IgA (T alpha) or IgE (T epsilon), respectively. The present study was undertaken to examine whether IgG- or IgM-secreting tumors influenced the frequency of T lymphocytes that express FcR for IgG or IgM. We studied mice bearing IgG- and IgM-secreting plasmacytomas and hybridomas. BALB/c mice injected subcutaneously with the IgG-secreting hybridoma HDP1 (gamma 1 kappa, anti-TNP) were sequentially examined for the frequencies and Lyt phenotypes of splenic lymphocytes bearing FcR for IgG (T gamma), IgM (T mu), and IgA (T alpha). A threefold increase in the frequency of T gamma lymphocytes that were Lyt-1-2+, L3T4- was seen. The frequencies of T mu and T alpha lymphocytes in these mice were not significantly altered. Similarly, mice injected subcutaneously with the IgM-secreting plasmacytoma MOPC 104E (mu lambda, anti-dextran) or the IgM-secreting hybridoma C1D1 (mu kappa, anti-ox RBC) were examined sequentially for the frequencies of T gamma, T mu, and T alpha lymphocytes. Mice with established IgM subcutaneous tumors showed a twofold increase in splenic, nylon wool-nonadherent T mu lymphocytes. This was associated with a relative increase in Lyt-2+ splenic T lymphocytes and a relative decrease in Lyt-1+ splenic T lymphocytes. No changes were observed in the frequencies of either T gamma or T alpha lymphocytes. These studies extend to IgG and IgM the observation that plasmacytomas and hybridomas secreting immunoglobulins of a specific isotype cause an expansion of T lymphocytes bearing FcR specific for the corresponding isotype. The expansion of FcR+ Lyt-1-2+ T lymphocytes likely represents an exaggerated, but otherwise normal, immunoregulatory response of the host. These cells may be an important element in the regulation of isotype expression.

A patient with chronic myelomonocytic leukemia with IgG K paraproteinemia, but no detectable plasmacytosis, is described. The patient was entering a blastic phase at the time of the most detailed studies. Cytological, cytochemical, and ultrastructural studies revealed a mixed myeloid proliferation with granulocytic forms predominating over monocytic elements. A variety of ultrastructural abnormalities, including defective granulation, was observed but no cells with highly developed rough endoplasmic reticulum were observed. Immunological marker studies showed that the mature myeloid cells possessed receptors for the Fc of IgG and weakly expressed the Ia-like P29/34 antigen. The mature myeloid cells also expressed both surface and intracytoplasmic Ig restricted to IgG K, and this IgG K persisted after 4 weeks in culture. A reverse plaque assay showed that the myeloid cells were capable of releasing IgG K in vitro, but studies involving the incorporation of radio-labeled amino acids showed no detectable Ig production by the myeloid cells. The possible interpretations of these data are discussed in some detail in relation to previous reports of paraproteinemia in myeloid proliferative disorders.

The effect of heat-aggregated immunoglobulin G's (HAgg-IgGs) on bacterial lipopolysaccharide- (LPS) induced polyclonal B cell activation (PBA) was studied. HAgg-IgGs (10 micrograms/ml or more) that were added to cultures of spleen cells obtained from BALB/c mice remarkably decreased the numbers of anti-trinitrophenyl (TNP) antibody-producing cells (APC) generated by LPS in cultures. This suppressive effect was seen when HAgg-IgGs were added within 1 hr after the initiation of culture. Later addition of HAgg-IgGs did not cause any effective suppression of the generation of APC. Moreover, the spleen cells, which had been pretreated with HAgg-IgGs, washed for removal of free HAgg-IgGs, and subsequently cultured, responded poorly to LPS. HAgg-IgG2b was more effective than HAgg-IgGs, but HAgg-IgG2a did not have any suppressive effect. A similar suppressive effect was observed when HAgg-IgG2b-Fc was added into culture, whereas none was noted with IgG2b-Fab. Indomethacin (IM), known as cyclooxygenase inhibitor, could completely abrogate the suppressive effects of HAgg-IgGs on LPS-induced PBA responses when it was added to the cell cultures together with HAgg-IgGs or to the cultures of HAgg-IgGs-pretreated cells. Addition of prostaglandin E2 (PGE2) showed strong suppression of the PBA responses of spleen cell cultures that had been pretreated with HAgg-IgGs and IM, but other PG analogues showed no suppression. Thus, it would appear that PGE2 has some role in the suppression of the LPS-induced PBA response.

In the present study we have identified and characterized three subpopulations of peripheral blood NK cells based on the surface expression of CD56 and CD16. We have designated these subsets CD16neg, CD16dim, and CD16bright according to the relative surface density of CD16. The CD16bright subset comprised about 10% to 15% of PBL, whereas the CD16dim and CD16neg subsets comprise less than 1% of the total lymphocytes. A detailed characterization of these subsets revealed both similarities and differences. The three subsets shared a great deal of phenotypic similarity, expressing CD2, CD7, CD11b, CD38, CD45R, CD18, and the p75 IL-2R on the majority of the cells in each subset. There were, however, several prominent phenotypic differences, particularly in the expression of CD57, CD11c, CD44, CD25, Leu-8, L263, and L265. The CD16neg cells were morphologically large agranular lymphocytes and demonstrated low levels of non-MHC restricted cytolysis of NK-sensitive tumor lines. The CD16dim and CD16bright subsets were large granular lymphocytes and revealed potent cytotoxicity against NK-sensitive targets. All subsets demonstrated IL-2-dependent activation and proliferation; however, the CD16dim and CD16neg subsets were preferentially responsive to very low concentrations of rIL-2. Although rIL-4 effectively inhibited the IL-2-induced cytolytic activation of all three NK cell subsets, only the CD16bright cells showed rIL-4 inhibition of IL-2 dependent proliferation. Cytokine transcription was also differentially regulated in the NK cell subsets after rIL-2 activation. Although TNF-alpha was equally transcribed in each subsets, IFN-gamma and serine protease-HF were preferentially transcribed in the CD16bright NK cells. Based on these results, we propose that these NK cell subsets represent portions of the NK cell differentiation pathway present in the peripheral blood.

The natural killer (NK) cell activity of human peripheral blood mononuclear cells (PBL) was found to be increased after incubation at 37 degrees C for 2 hr. The observed increase was shown to be associated with release from inhibition by human serum factors, because incubation in autologous serum interfered with augmentation. The serum-mediated effect appeared attributable to the degree of binding of labile IgG to PBL and could be reduced by selective depletion of IgG from the serum. Human monomeric IgG was found to efficiently inhibit the culture-induced augmentation of NK activity; the inhibitory IgG had properties consistent with those described for cytophilic IgG and was mediated through the Fc region of IgG. The inhibition by monomeric IgG occurred at 0 degrees C as well as at 37 degrees C and this could be induced even after culture-induced augmentation of NK activity. Thus, binding of monomeric IgG to human PBL appears to reversibly inhibit their NK activity. These results provide evidence for a novel mechanism for negative regulation of NK activity.

Surface-bound platelet IgG and IgM were measured by an enzyme-linked immunosorbent assay (ELISA) using washed platelets and commercially available alkaline phosphatase anti-human immunoglobulins (Fc-specific). With this technique platelets from normal donors had small amounts of platelet-bound IgG ranging from 0.00 to 0.16 A405 (absorbance at 405 nm wavelength) (10(7) platelets)-1 (0 to 124 ng) and of platelet-bound IgM ranging from 0.00 to 0.05 A405 (10(7) platelets)-1. Eight out of 10 (80%) thrombocytopenic patients with idiopathic autoimmune thrombocytopenic purpura (IATP) had values of both IgG and IgM exceeding the normal range. In addition, one patient (8%) had platelet-bound IgM only. An inverse relationship was demonstrated in patients with IATP between the blood platelet count and the amount of both IgG and IgM. Increased values were also demonstrated in patients with SLE and patients with monoclonal hypergammaglobulinaemia. The direct ELISA is a useful and reproducible technique for platelet-bound IgG and IgM, which requires standard laboratory equipment only.

Suspensions of human platelets were incubated with various immunoglobulin preparations and subsequently stained with FITC-conjugated antisera. Incubation with monomeric, IgG, but not with monomeric IgM, IgA, IgD nor with IgE, gave a positive staining of the platelets. Incubation of platelets with monomeric IgG1 and IgG3 as well as with Fc and pFc'-fragments from IgG3 also gave a positive staining while incubation with monomeric IgG2 and IgG4 did not. Thus, IgG binds to Fc receptors on the surface of human platelets with the CH3 domain of the Fc region. Heat aggregation also caused binding of IgG2 but not with IgG4 proteins to human platelet Fc receptors. The majority of platelet preparations both from ITP and SLE patients gave a positive staining using direct immunofluorescence technique. Incubation of normal human platelets with sera from ITP and SLE patients gave a strong surface staining of normal platelets. Strong staining was also obtained with pepsin-digested sera from most patients with ITP while pepsin-digested sera from patients with SLE did not give a positive staining. It is therefore concluded that the majority of sera from patients with ITP contain antibodies with specificities for platelet surface antigens while sera from patients with SLE contain immune complexes that react with platelet Fc receptors through the Fc parts of the IgG antibodies.

In order to verify the hypothesis of the presence of IgM (or an IgM-like substance) capable of inhibiting thyroid hormone binding to serum proteins and, therefore, capable of enhancing serum free thyroid fractions in non-thyroid illnesses (NTI), we measured TBG and TBPA maximum binding capacities and TBG concentration by an immunoradiometric system in normal pooled sera (NPS) before and after enrichment with purified immunoglobulins (IgM and IgG) from euthyroid NTI sera (free T4, free T3 and TSH levels were normal). A known amount of TBG was diluted 1:2-1:6 with deionized water or with IgM from NPS or from each of 6 NTI sera; the measured values were not different from these expected on theoretical grounds. Likewise, IgM or IgG from normal or from each of 7 NTI sera failed to modify both TBG and TBPA capacities of different NPS, and NTI immunoglobulins showed no binding activity directed to [125I]T4, [125I]T3 or [125I]TBG. In addition, no inhibitory influence of TBG and TBPA capacities was observed when the whole euglobulin fraction obtained by precipitating the same 7 NTI sera by PEG was mixed with NPS. On the other hand, a significant IgM inhibitory effect on the binding of labelled T4 to TBG was found, only when IgM concentrations were experimentally rendered 41 times greater than that requested in the working mixtures. We conclude that no immunoglobulin inhibitor of thyroid hormone binding to transport proteins was evident in the NTI sera investigated.