RESUMO
The human Ab response to many common pathogens is oligoclonal, with restricted usage of Ig V-genes. Intriguingly, the IGVK3-11 and IGVH3-30 V-genes are repeatedly paired in protective Abs against the 23F polysaccharide of Streptococcus pneumoniae, as well as against the gB envelope protein of human CMV, where germline-encoded amino acids make key contacts with the gB protein. We constructed IgGs encoded by the germline IGVK3-11 and IGVH3-30 V-genes together with DNA encoding the respective CDR3 regions of the L chain and H chain found in a hypermutated anti-23F Ab. These IgGs encoded by germline V-genes bound specifically to 23F pneumococcal capsular polysaccharides with no reactivity to other serotypes of pneumococcal capsular polysaccharides or arrayed glycans and recognized L-rhamnose, a component of the 23F repeating subunit. IgGs encoded by this pair of germline V-genes mediated complement-dependent phagocytosis of encapsulated 23F S. pneumoniae by human neutrophils. Mutations in CDRL3 and CDRH3 had significant effects on binding. Thus, IGKV3-11 and IGHV3-30, depending on with which distinct DNA sequences encoding CDR3 they are recombined, can encode binding sites for protective Abs against chemically distinct Ags and thus, may encode innate immunological memory against human CMV and S. pneumoniae.
Assuntos
Regiões Determinantes de Complementaridade/genética , Imunidade Inata/genética , Região Variável de Imunoglobulina/genética , Memória Imunológica/genética , Vírus Sinciciais Respiratórios/imunologia , Hipermutação Somática de Imunoglobulina/genética , Streptococcus pneumoniae/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Sítios de Ligação de Anticorpos/genética , Células Cultivadas , Regiões Determinantes de Complementaridade/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/metabolismo , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/metabolismo , Dados de Sequência Molecular , Neutrófilos/imunologia , Neutrófilos/microbiologia , Neutrófilos/virologia , Vírus Sinciciais Respiratórios/metabolismo , Vírus Sinciciais Respiratórios/patogenicidade , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/patogenicidadeRESUMO
The exceptional value of gene targeting technology to generate mouse models of human disease exists under the shadow of potential genetic errors. We previously observed an unexpected brain-behavior phenotype that resulted from a gene-targeting experiment designed to delete the Zfa gene. Given that the transcription of Zfa is restricted to the germ cell lineage of adult testis, it was both a surprise and a concern when the resulting mice had a phenotype present in both sexes that included abnormal brains and violent behavior. We hypothesized that an unrelated mutation may have been responsible for the unexpected phenotype. Here we show that the single gene mutation, Nr2e1(frc) (fierce), which was responsible for the brain-behavior phenotype, existed in the embryonic stem (ES) cell even before the derivation of the Zfa knockout mice. Our work thus highlights a concern in gene targeting, namely, that ES cells can harbor unexpected mutations, which can lead to genotype-phenotype misattribution. Based on our findings, we caution the gene-targeting community to use low-passage ES cells, to characterize mice derived from more than one independently targeted ES cell clone, and to backcross mice to allow for segregation of distant but linked mutations.
