Molecular analysis of IgE H-chain transcripts expressed in vivo by peripheral blood lymphocytes from normal and atopic individuals.
The low levels of IgE produced by PBMC from normal individuals has so far prevented an analysis of their IgE H chain repertoire. Using a nested polymerase chain reaction approach, we were able to detect epsilon transcripts in all normal and allergic individuals we investigated. We further cloned epsilon CDR3/FW4 regions from two normal and two atopic individuals with low serum IgE levels. Sequence analysis of 104 clones identified 26 different epsilon CDR3/FW4 regions and an additional number of clonally related transcripts in the two atopic individuals. Preferential usage of DH genes from the DXP family (33%) and of the JH4b gene (35%) were observed, similar to reported findings for the IgM-producing peripheral blood B cell subset. Using CDR3 specific oligonucleotides, we detected the CDR3/FW4 regions of a particular set of clonally related epsilon transcripts in mu and gamma 4 transcripts of the same individual. This finding demonstrates the in vivo production of IgE together with the two other Ig isotypes (IgM and IgG4) by the progeny of a common B cell precursor, and suggests a possible mechanism for regulating the allergic response. The clonally related epsilon transcripts were found to be only of the secreted form. We give also evidence that the IgE-producing B cells undergo somatic mutation because a number of identical mutations were observed in the FW4 regions of epsilon and mu clonally related transcripts. Some of these mutations were shared with other transcripts from the same and other individuals, supporting the existence of sequence-specific hot spots for the somatic hyper-mutation machinery in the JH gene segments.
Efremov
D G
DG
Molecular Immunology Group, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
Batista
F D
FD
Burrone
O R
OR
D000328:Adult; D001483:Base Sequence; D003001:Cloning, Molecular; D015870:Gene Expression; D005803:Genes, Immunoglobulin; D006801:Humans; D006967:Hypersensitivity / Q000378:metabolism*; D007127:Immunoglobulin Constant Regions / Q000235:genetics; D007074:Immunoglobulin G / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007139:Immunoglobulin epsilon-Chains / Q000235:genetics*; D007142:Immunoglobulin gamma-Chains / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics
D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D007139:Immunoglobulin epsilon-Chains; D007142:Immunoglobulin gamma-Chains; D008562:Membrane Glycoproteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger