RESUMO
The transfer of maternal gamma-globulin (IgG) provides the neonate with humoral immunity during early life. In humans, maternal IgG is transported across the placenta during the third trimester of pregnancy. The expression of the MHC class I-related receptor, FcRn, in the human placenta suggests that this Fc receptor might be involved in the delivery of maternal IgG, but direct evidence to support this is lacking. In the current study an ex vivo placental model has been used to analyze the maternofetal transfer of a recombinant, humanized (IgG1) antibody in which His435 has been mutated to alanine (H435A). In vitro binding studies using surface plasmon resonance indicate that the mutation ablates binding of the antibody to recombinant mouse and human FcRn. Relative to the wild-type antibody, the H435A mutant is deficient in transfer across the placenta. Significantly, the mutation does not affect binding to Fc gamma RIII, an FcR that has been suggested in earlier studies to mediate the transfer of maternal IgG. The analyses demonstrate that binding of an IgG to FcRn is a prerequisite for transport across the perfused placenta. FcRn therefore plays a central role in the maternofetal delivery of IgG and this has implications for the use of protein engineering to improve the properties of therapeutic antibodies.
Assuntos
Antígenos de Histocompatibilidade Classe I/fisiologia , Imunidade Materno-Adquirida/imunologia , Imunoglobulina G/metabolismo , Troca Materno-Fetal/imunologia , Receptores Fc/fisiologia , Animais , Sítios de Ligação de Anticorpos/genética , Sítios de Ligação de Anticorpos/imunologia , Feminino , Meia-Vida , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Imunoglobulina G/genética , Técnicas In Vitro , Camundongos , Mutação/genética , Placenta/imunologia , Placenta/metabolismo , Plasmídeos/biossíntese , Plasmídeos/imunologia , Gravidez , Receptores Fc/sangue , Receptores de IgG/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Myelin basic protein (MBP)-specific T cells play a critical role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a prototype for T cell-mediated autoimmunity. In PL/J and B10.PL mice (H-2(u) haplotype), the immunodominant epitope of MBP is represented by an N-terminal nonameric peptide, MBP1-9. To date, the MBP1-9-specific T cell repertoire has not been analyzed in quantitative terms. In the present study we demonstrate, using MHC class II tetramers, that 15,000-70,000 self-antigen-specific T(h) cells accumulate in the draining lymph nodes following immunization with spinal cord homogenate or MBP1-9. In contrast, MBP1-9-specific T cells are undetectable in unimmunized H-2(u) mice and represent >60% of the CD4 cells in naive mice transgenic for a TCR specific for this epitope. The results suggest that the extremely low affinity of the N-terminal peptide for I-A(u) does not limit the MBP1-9-specific T cells from expanding into a sizeable pool of autoreactive T cells. Therefore, the primary immune response to MBP1-9 does not differ quantitatively from previously reported CD4(+) T cell responses to foreign antigens.
Assuntos
Autoantígenos/imunologia , Antígenos H-2/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Fragmentos de Peptídeos/imunologia , Linfócitos T/imunologia , Animais , Autoantígenos/administração & dosagem , Epitopos de Linfócito T/biossíntese , Epitopos de Linfócito T/imunologia , Adjuvante de Freund/imunologia , Antígenos H-2/biossíntese , Antígenos de Histocompatibilidade Classe II/biossíntese , Antígenos de Histocompatibilidade Classe II/química , Hibridomas , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Básica da Mielina/administração & dosagem , Proteína Básica da Mielina/imunologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/biossínteseRESUMO
The analysis of the pharmacokinetics of wild-type and mutated Fc fragments derived from human IgG1 indicates that Ile253, His310 and His435 play a central role in regulating serum half-life in mice. Reduced serum half-life of the recombinant, mutated fragments correlates with decreased binding to the MHC class I-related neonatal Fc receptor, FcRn. In addition, the analysis of an Fc fragment in which His435 is mutated to Arg435 demonstrates that the sequence difference at this position between human IgG1 (His435) and IgG3 (Arg435) most likely accounts for the shorter serum half-life of IgG3 relative to IgG1. In contrast to His310 and His435, the data indicate that His433 does not play a role in regulating the serum half-life of human IgG1. Thus, the interaction site of mouse FcRn on human and mouse IgG1 involves the same conserved amino acids located at the CH2-CH3 domain interface of the IgG molecule. The sequence similarities between mouse and human FcRn suggest that these studies have direct relevance to understanding the factors that govern the pharmacokinetics of therapeutic IgG.