Dynamics of spontaneous in vitro isotype switching in hybridoma cells

Abstract

Mouse immunoglobulin heavy chain constant regions (C{dollar}\sb{lcub}\rm H{rcub}{dollar}) are encoded by eight genes that are downstream from the variables region (V{dollar}\sb{lcub}\rm H{rcub}{dollar}) genes and have the following order: C{dollar}\sb{lcub}\mu{rcub}{dollar}, C{dollar}\sb{lcub}\delta{rcub}{dollar}, C{dollar}\sb{lcub}\gamma{rcub}{dollar}1, C{dollar}\sb{lcub}\gamma{rcub}{dollar}3, C{dollar}\sb{lcub}\gamma{rcub}{dollar}2b, C{dollar}\sb{lcub}\gamma{rcub}{dollar}2a, C{dollar}\sb{lcub}\varepsilon{rcub}{dollar} and C{dollar}\sb{lcub}\alpha{rcub}{dollar}. They define different isotypes and encode molecules with different biological functions. During B cell differentiation, the upstream V region can be expressed with any of the isotypes by successive recombination steps that place the V{dollar}\sb{lcub}\rm H{rcub}{dollar} gene upstream from each C{dollar}\sb{lcub}\rm H{rcub}{dollar} gene. This isotype switch process occurs at a high frequency in vivo and may be mediated by switch regions that are reiterated sequences upstream of each C{dollar}\sb{lcub}\rm H{rcub}{dollar} gene.;Isotype switching also occurs in cultured antibody cells, but at lower frequencies than in vivo. I have used a sib selection technique to generate families of class switch variants from anti PC and anti-ricin IgM and IgG producing hybridomas. The different classes and subclasses of immunoglobulin produced have the same antigen binding properties as the parental antibody.;A detailed analysis of switch frequencies was done for two anti-PC IgM producing hybridomas. These include analysis of primary switches from the parental IgM producing hybridomas, and secondary switches from three independent IgG1 switch variants derived from one of the IgM parentals (PC1).;The frequencies observed were low, ranging from 0.2 to 1.0 {dollar}\times{dollar} 10{dollar}\sp{lcub}-6{rcub}{dollar} for primary IgM to IgG switches and 0.5 to 12.0 {dollar}\times{dollar} 10{dollar}\sp{lcub}-6{rcub}{dollar} for secondary switches from one IgG1 subclass to another. Southern blot analysis indicated that most of the recombination events occurred within switch regions. Furthermore, the frequency of switching from IgM to IgG correlated with the amount of {dollar}\mu{dollar} switch region retained in the cells. For secondary switch events the configuration of the switch regions upstream of the C{dollar}\sb{lcub}\gamma{rcub}{dollar}1, created by the primary switch event, determined the frequency with which switches occurred from {dollar}\sb{lcub}\gamma{rcub}{dollar}1 to the downstream constant regions.