Somatic mutation of immunoglobulin V(H)6 genes in human infants - PubMed (original) (raw)

Somatic mutation of immunoglobulin V(H)6 genes in human infants

J Ridings et al. Clin Exp Immunol. 1998 Oct.

Abstract

Infants respond to antigen by making antibody that is generally of low affinity for antigen. Somatic hypermutation of immunoglobulin genes, and selection of cells expressing mutations with improved affinity for antigen, are the molecular and cellular processes underlying the maturation of antibody affinity. We have reported previously that neonates and infants up to 2 months of age, including individuals undergoing strong immunological challenge, show very few mutated V(H)6 sequences, with low mutation frequencies in mutated sequences, and little evidence of selection. We have now examined immunoglobulin genes from healthy infants between 2 and 10 months old for mutation and evidence of selection. In this age group, the proportion of V(H)6 sequences which are mutated and the mutation frequency in mutated sequences increase with age. There is evidence of selection from 6 months old. These results indicate that the process of affinity maturation, which depends on cognate T-B cell interaction and functional germinal centres, is approaching maturity from 6 months old.

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Figures

Fig. 2

Summary of heteroduplex analysis of VH6 sequences isolated from cloned samples. Each bar represents the percentage of mutated sequences detected for each sample. The total number of VH6 sequences analysed is given above each bar. The VH6 control line represents the proportion of mutated sequences which arise from polymerase chain reaction (PCR) amplification, and is the mean value from four separate cloning reactions of a germ-line VH6–D–J rearrangement and the subsequent screening of 125 VH6+ clones. Sequential blood samples taken from the same donor are shown by † or *.

Fig. 1

Heteroduplex analysis of VH6+ clones isolated from infant peripheral blood leucocytes (PBL). Mutated clones were identified by heteroduplex formation (lanes 3, 9, 10, 11, 12, 14) between the unknown VH6 sequence and a known germ-line VH6 sequence (run unmixed in lane 15). Mutations were confirmed by sequencing. Heteroduplex negative clones were considered unmutated (lanes 1, 2, 4, 5, 6, 7, 8, 13), and some of these were also sequenced to confirm this. A positive control (lane 16) of germ-line mixed with mutated VH6 sequence (two mutations) showed a double band. M, Marker lane (100-bp ladder; Promega).

Fig. 3

Sequence analysis of the mutated VH6 genes isolated from infant and adult peripheral blood leucocytes (PBL). Each bar represents the mean mutation frequency, for the mutated sequences, for a particular sample. Mutation frequency is expressed as the mean number of mutations per 100 bp of sequence. The numbers above the bar represent the number of unique mutated sequences isolated for each sample. The VH6 control line represents the mean mutation frequency from mutated clones generated by the polymerase chain reaction (PCR) protocol. Sequential blood samples taken from the same donor are shown by † or *. We were unable to obtain satisfactory sequencing data for those mutated clones detected in infants aged 62 and 131 days, hence no mutation frequency is shown for these samples.

Fig. 4

Schematic representation of mutations in rearranged VH6 sequences isolated from infants and adult blood samples. Each line represents one mutated VH6 sequence. The age of each infant is given in days on the left, with symbols denoting clonal families within each sample. The CDR and framework regions (FWR) are indicated and mutations are represented by different symbols: ▴, replacement mutations; I, silent mutations; S, stop codon; −, single base pair deletion. Functional sequences are represented by + and non-functional sequences arising from an altered reading frame or stop codons are shown as −.

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Somatic mutation of immunoglobulin V(H)6 genes in human infants - PubMed (original) (raw)