RESUMEN
One important function of humoral immunity is toxin neutralization. The current view posits that neutralization results from antibody-mediated interference with the binding of toxins to their targets, a phenomenon viewed as dependent only on antibody specificity. To investigate the role of antibody constant region function in toxin neutralization, we generated IgG2a and IgG2b variants of the Bacillus anthracis protective antigen-binding IgG1 monoclonal antibody (mAb) 19D9. These antibodies express identical variable regions and display the same specificity. The efficacy of antibody-mediated neutralization was IgG2a > IgG2b > IgG1, and neutralization activity required competent Fcγ receptor (FcγR). The IgG2a mAb prevented lethal toxin cell killing and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase cleavage more efficiently than the IgG1 mAb. Passive immunization with IgG1 and IgG2a mAb protected wild-type mice, but not FcγR-deficient mice, against B. anthracis infection. These results establish that constant region isotype influences toxin neutralization efficacy of certain antibodies through a mechanism that requires engagement of FcγR. These findings highlight a new parameter for evaluating vaccine responses and the possibility of harnessing optimal FcγR interactions in the design of passive immunization strategies.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Bacillus anthracis/inmunología , Toxinas Bacterianas/inmunología , Inmunoglobulina G/inmunología , Receptores de IgG/inmunología , Animales , Vacunas contra el Carbunco/inmunología , Anticuerpos Antibacterianos , Línea Celular , Inmunización Pasiva , Ratones , Ratones Noqueados , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/inmunología , Receptores de IgG/genéticaRESUMEN
Replacement of aspartic acid by alanine at position 265 (D265A) in mouse IgG1 results in a complete loss of interaction between this isotype and low-affinity IgG Fc receptors (Fc gammaRIIB and Fc gammaRIII). However, it has not yet been defined whether the D265A substitution could exhibit similar effects on the interaction with two other Fc gammaR (Fc gammaRI and Fc gammaRIV) and on the activation of complement. To address this question, 34-3C anti-RBC IgG2a and IgG2b switch variants bearing the D265A mutation were generated, and their effector functions and in vivo pathogenicity were compared with those of the respective wild-type Abs. The introduction of the D265A mutation almost completely abolished the binding of 34-3C IgG2a and IgG2b to all four classes of Fc gammaR and the activation of complement. Consequently, these mutants were hardly pathogenic. Although oligosaccharide side chains of these mutants were found to contain higher levels of sialic acids than those of wild-type Abs, the analysis of enzymatically desialylated D265A variants ruled out the possibility that very poor Fc-associated effector functions of the D265A mutants were due to an increased level of the mutant Fc sialylation. Thus, our results demonstrate that aspartic acid at position 265 is a residue critically implicated in triggering the Fc-associated effector functions of IgG, probably by defining a crucial three-dimensional structure of the Fc region.
Asunto(s)
Ácido Aspártico/fisiología , Activación de Complemento/inmunología , Inmunoglobulina G/metabolismo , Alanina/genética , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/fisiología , Anemia Hemolítica Autoinmune/genética , Anemia Hemolítica Autoinmune/inmunología , Animales , Anticuerpos Monoclonales/toxicidad , Ácido Aspártico/genética , Autoanticuerpos/toxicidad , Activación de Complemento/genética , Eritrocitos/inmunología , Inmunoglobulina G/química , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Isoformas de Proteínas/química , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , Receptores Fc/química , Receptores Fc/deficiencia , Receptores Fc/genética , Receptores Fc/fisiología , Ácidos Siálicos/genética , Relación Estructura-ActividadRESUMEN
Four murine IgG subclasses display markedly different Fc-associated effector functions because of their differential binding to three activating IgG Fc receptors (FcgammaRI, FcgammaRIII, and FcgammaRIV) and C1q. Previous analysis of IgG subclass switch variants of 34-3C anti-RBC monoclonal autoantibodies revealed that the IgG1 subclass, which binds only to FcgammaRIII and fails to activate complement, displayed the poorest pathogenic potential. This could be related to the presence of a three amino acid deletion at positions 233-235 in the CH2 domain uniquely found in this subclass. To address this question, IgG1 insertion and IgG2b deletion mutants at positions 233-235 of 34-3C anti-RBC Abs were generated, and their ability to initiate effector functions and their pathogenicity were compared with those of the respective wild-type Abs. The insertion of amino acid residues at positions 233-235 enabled the IgG1 subclass to bind FcgammaRIV but did not improve the binding to C1q. Accordingly, its pathogenicity was enhanced but still inferior to that of IgG2b. In contrast, the IgG2b deletion mutant lost its ability to bind to FcgammaRIV and activate complement. Consequently, its pathogenicity was markedly diminished to a level comparable to that of IgG1. Our results demonstrated that the initiation of FcgammaR- and complement-mediated effector functions of IgG2b was profoundly affected by the three amino acid deletion at positions 233-235, but that this natural three amino acid deletion could only partially explain the poor binding of IgG1 to FcgammaRIV and C1q. This indicates the lack in the IgG1 subclass of as yet unknown motifs promoting efficient interaction with FcgammaRIV and C1q.