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The Influence of Human IgG Subclass and Allotype on Complement Activation.

ABSTRACT: Complement activation via the classical pathway is initiated when oligomeric Igs on target surfaces are recognized by C1 of the complement cascade. The strength of this interaction and activation of the complement system are influenced by structural variation of the Ab, including Ab isotype, subclass, and glycosylation profile. Polymorphic variants of IgG have also been described to influence Fc-dependent effector functions. Therefore, we assessed complement binding, deposition, and complement-dependent cytotoxicity (CDC) of 27 known IgG allotypes with anti-trinitrophenyl specificity. Differences between allotypes within subclasses were minor for IgG1, IgG3, and IgG4 allotypes, and more substantial for IgG2. Allelic variant IGHG2*06, containing a unique serine at position 378 in the CH3 domain, showed less efficient complement activation and CDC compared with other IgG2 polymorphisms. We also observed variable cell lysis between IgG1 and IgG3, with IgG3 being superior in lysis of human RBCs and Ramos cells, and IgG1 being superior in lysis of Raji and Wien133 cells, demonstrating that a long-standing conundrum in the literature depends on cellular context. Furthermore, we compared IgG1 and IgG3 under different circumstances, showing that Ag density and Ab hinge length, but not complement regulators, define the context dependency of Ab-mediated CDC activity. Our results point toward a variation in the capacity of IgG subclasses to activate complement due to single amino acid changes and hinge length differences of allotypes to activate complement, which might give new insights on susceptibility to infectious, alloimmune, or autoimmune diseases and aid the design of Ab-based therapeutics.

SUBMITTER: Damelang T

PROVIDER: S-EPMC10656437 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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The Influence of Human IgG Subclass and Allotype on Complement Activation.

Damelang Timon T de Taeye Steven W SW Rentenaar Rosa R Roya-Kouchaki Kasra K de Boer Esther E Derksen Ninotska I L NIL van Kessel Kok K Lissenberg-Thunnissen Suzanne S Rooijakkers Suzan H M SHM Jongerius Ilse I Mebius Mirjam M MM Schuurman Janine J Labrijn Aran F AF Vidarsson Gestur G Rispens Theo T

Journal of immunology (Baltimore, Md. : 1950) 20231201 11

Complement activation via the classical pathway is initiated when oligomeric Igs on target surfaces are recognized by C1 of the complement cascade. The strength of this interaction and activation of the complement system are influenced by structural variation of the Ab, including Ab isotype, subclass, and glycosylation profile. Polymorphic variants of IgG have also been described to influence Fc-dependent effector functions. Therefore, we assessed complement binding, deposition, and complement-d ...[more]

PMID: 37843500

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Project description:T cell redirection mediated by bispecific antibodies (BsAbs) is a promising cancer therapy. Dual antigen binding is necessary for potent T cell redirection and is influenced by the structural characteristics of a BsAb, which are dependent on its IgG subclass. In this study, model BsAbs targeting CD19xCD3 were generated in variants of IgG1, IgG2, and IgG4 carrying Fc mutations that reduce FcγR interaction, and two chimeric IgG subclasses termed IgG1:2 and IgG4:2, in which the IgG1- or IgG4-F(ab)2 are grafted on an IgG2 Fc. Molecules containing an IgG2 or IgG4-F(ab)2 domain were confirmed to be the most structurally compact molecules. All BsAbs were shown to bind both of their target proteins (and corresponding cells) equally well. However, CD19xCD3 IgG2 did not bind both antigens simultaneously as measured by the absence of cellular clustering of T cells with target cells. This translated to a reduced potency of IgG2 BsAbs in T-cell redirection assays. The activity of IgG2 BsAbs was fully restored in the chimeric subclasses IgG4:2 and IgG1:2. This confirmed the major contribution of the F(ab)2 region to the BsAb's functional activity and demonstrated that function of BsAbs can be modulated by engineering molecules combining different Fc and F(ab)2 domains. Abbreviations: ADCC: Antibody-dependent cellular cytotoxicity; AlphaScreenTM: Amplified Luminescent Proximity Homogeneous Assay Screening; ANOVA: Analysis of variance; BiTE: bispecific T-cell engager; BSA: bovine serum albumin; BsAb: bispecific antibody; cFAE: controlled Fab-arm exchange; CDC: complement-dependent cellular cytotoxicity; CIEX: cation-exchange; CIR: chimeric immune receptor; DPBS: Dulbecco's phosphate-buffered saline; EC50 value: effective concentration to reach half-maximum effect; EGFR: epidermal growth factor receptor; EI: expansion index (RAt=x/RAt=0); FACS: fluorescence-activated cell sorting; FVD: fixable viability dye; HI-HPLC: hydrophobic interaction HPLC; HI-FBS: heat-inactivated fetal bovine serum; HPLC: high-pressure liquid chromatography; IC50 value: effective concentration to reach half-maximum inhibition; IQ: Inhibition Quotient; IS: immunological synapse; MES: 2-(N-morpholino)ethanesulfonic acid; R-PE: recombinant phycoerythrin; RA: red area in μm2/well; RD: receptor density; RFP: red fluorescent protein; Rg: radius of gyration; RSV: respiratory syncytial virus; SAXS: small-angle x-ray scattering; scFv: single-chain variable fragment; SD: standard deviation; SPR: surface plasmon resonance; WT: wild-type.

Project description:Autoimmune neuropathy associated with antibodies against pan-neurofascin is a new subtype of nodo-paranodopathy. It is relevant because it is associated with high morbidity and mortality. Affected patients often require intensive care unit treatment for several months, and data on the reversibility and long-term prognosis are limited. The pathogenicity including IgG subclass-associated mechanisms has not been unravelled, nor directly compared to anti-neurofascin-155 IgG4-related pathology. Understanding the underlying pathology might have a direct impact on treatment of these severely affected patients. By a multicentre combined prospective and retrospective approach, we provide clinical data of a large cohort of patients with anti-neurofascin-associated neuropathy (n = 18) including longitudinal titre and neurofilament light chain assessment via Ella® and relate clinical data to in vitro pathogenicity studies of anti-neurofascin antibodies. We assessed antibody binding characteristics and the pathogenic effects of anti-pan-neurofascin versus neurofascin-155 antibodies on living myelinating dorsal root ganglia co-cultures. Additionally, we analysed the IgG subclass profile and the complement binding capacity and effector functions considering the effects of intravenous immunoglobulin preparations via enzyme-linked immunosorbent and cell-based assays. In contrast to chronic neurofascin-155 IgG4-associated neuropathy, anti-pan-neurofascin-associated disease presented with a high morbidity and mortality, but as a monophasic and potentially reversible disorder. During follow-up, antibodies were no longer detectable in 8 of 11 patients. Anti-pan-neurofascin had direct access to the nodes of Ranvier in myelinating cultures titre-dependently, most probably inducing this severe phenotype. Antibody preincubation led to impaired paranode formation, destruction of paranodal architecture and alterations on paranodal myelin and sensory neurons in the cultures, with more severe effects than neurofascin-155 antibodies. Besides IgG4, subclass IgG3 was detected and associated with complement binding and cytotoxic effects in vitro. As a possible correlate of axonal damage in vivo, we detected highly increased serum neurofilament light chain levels (sNF-L), correlating to serum C3a. Still, sNF-L was not identified as a marker for poor prognosis, but rather as an intra- and interindividual marker for acuteness, severity and course, with a strong decrease during recovery. Our data provide evidence that anti-pan-neurofascin antibodies directly attack the node and induce severe and acute, but potentially reversible, nodo-paranodal pathology, possibly involving complement-mediated mechanisms. Screening for autoantibodies thus is crucial to identify this subset of patients who benefit from early antibody-depleting therapy. Titre and sNF-L might serve as valuable follow-up parameters. The prospect of a favourable outcome has high relevance for physicians, patients and relatives during months of critical care.

Project description:BackgroundFactors associated with IgG levels in adults with IgG subclass deficiency (IgGSD) are incompletely understood. We studied adults with IgGSD with subnormal IgG1 only, subnormal IgG1/IgG3, or subnormal IgG3 only without other subnormal IgG subclasses, IgA, or IgM. We compiled: age; sex; autoimmune condition(s) (AC); atopy; IgG, IgG subclasses, IgA, IgM; IgGsum (IgG1 + IgG2 + IgG3 + IgG4); and D (percentage difference between IgGsum and IgG). We compared attributes of patients with/without subnormal IgG (< 7.00 g/L; subnormal IgG1 subclass groups only) and analyzed IgGsum and IgG relationships. We performed backward stepwise regressions on IgG using independent variables IgG subclasses, age, and sex and on D using independent variables age and sex.ResultsThere were 39 patients with subnormal IgG1 only (89.7% women), 53 with subnormal IgG1/IgG3 (88.7% women), and 115 with subnormal IgG3 only (91.3% women). Fifteen patients (38.5%) and 32 patients (60.4%) in the respective subnormal IgG1 subclass groups had subnormal IgG. Attributes of patients with/without IgG < 7.00 g/L were similar, except that AC prevalence was lower in patients with subnormal IgG1 only and IgG < 7.00 g/L than ≥ 7.00 g/L (p = 0.0484). Mean/median IgG1 and IgG2 were significantly lower in patients with IgG < 7.00 g/L in both subnormal IgG1 subclass groups (p < 0.0001, all comparisons). Regressions on IgG in three subclass groups revealed positive associations with IgG1 and IgG2 (p < 0.0001 each association). Regressions on D revealed no significant association. IgG1 percentages of IgGsum were lower and IgG2 percentages were higher in patients with subnormal IgG1 subclass levels than subnormal IgG3 only (p < 0.0001 all comparisons).ConclusionsWe conclude that both IgG1 and IgG2 are major determinants of IgG in patients with subnormal IgG1, combined subnormal IgG1/IgG3, or subnormal IgG3 and that in patients with subnormal IgG1 or combined subnormal IgG1/IgG3, median IgG2 levels are significantly lower in those with IgG < 7.00 g/L than those with IgG ≥ 7.00 g/L.

Dataset Information

The Influence of Human IgG Subclass and Allotype on Complement Activation.

Publications

The Influence of Human IgG Subclass and Allotype on Complement Activation.

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