Isotype switching and somatic hypermutations of immunoglobulin heavy chain in cultured B cell lines

Abstract

Somatic hypermutation and isotype switching of immunoglobulin genes are important parts of the immune response. The molecular correlates of switching were compared in switch variants of two hybridoma lines that spontaneously switch from {dollar}\gamma{dollar}1 to either {dollar}\gamma{dollar}2a and {dollar}\gamma{dollar}2b at high and low rates. Correlation between levels of I{dollar}\gamma{dollar}2a germline transcripts and switching rates was observed in the 36.65 subclones but not in the PC1.4.1 subclones. High switching variants of both 36.65 and PC1.4.1 had higher recombination activities than low switching subclones.;Somatic mutation was also investigated. We have stably transfected B cell lines with {dollar}\gamma{dollar}2a heavy chain constructs containing a TAG nonsense codon in their V regions that is either part of the putative (T)AGC hot spot or the (T)AGA non-hot spot motif. In the NSO plasma cell line, the (T)AGC hot spot motif mutates at rates of {dollar}{lcub}\sim{rcub}6 \times 10\sp{lcub}-4{rcub}{dollar}/bp/generation and {dollar}{lcub}\sim{rcub}3 \times 10\sp{lcub}-5{rcub}{dollar}/bp/generation at residues 38 and 94 in the V region. At each of these locations, the mutation rates of the (T)AGC hot spot motif is 20-30 times higher than the (T)AGA non-hot spot motif. However, the AGA non-hot spot motif mutates at as high a rate as the hot spot motif when it is located adjacent to hot spot motifs, suggesting that more extended sequences influence susceptibility to mutation.;To determine the role of intronic enhancer E{dollar}\mu{dollar} in V-region hypermutation, E{dollar}\mu{dollar} in the {dollar}\gamma{dollar}2a construct was replaced with either the CMV or SV40 enhancer. These constructs mutated at similar high rates in NSO, indicating that E{dollar}\gamma{dollar} is not specifically required for IgH mutation in this system. Much lower mutation rates were obtained from transfection of these constructs into CHO and fibroblast non-B cell lines, documenting B cell specificity of hypermutation in this system.;A multiple mutation accumulation model was proposed for determining mutation rates in vitro and in vivo, and mutation probability. A new model for somatic mutation was also proposed.