Specific V gene usage in anti-phosphorylcholine ige antibodies

Abstract

Three hybridomas from phosphorylcholine(PC)-KLH immunized BALB/c mice producing IgE antibodies against the PC hapten were investigated for their fine specificity to the hapten and usage of V gene segments in H- and L-chains. All three IgE antibodies recognize the entire azophenyl-PC hapten. They are T15 Id negative and do not bind to the natural PC determinant expressed by the Streptococcus carbohydrate R36A. T15 Id positive IgE antibodies could neither be elicited by immunization in detectable amounts nor generated by the cell fusion technique. By using the Southern blot technique and nucleotide sequence analysis of PCR amplified V HDJ H and V LJ L rearrangements, we have demonstrated that the three IgE anti-PC hybridomas use the V H1-DSP2-J H2, the V HOX1-DSP2-J H3 or the V H36-60-D-J H2 gene segment combinations for the H chain together with the V κ1C-J κ1, V κlC-J gk2 or V γl-J γl genes for the L chains. Except for the V H36–60, the same gene segments were found in different combinations in anti-PC antibodies of other Ig classes than IgE. However, high rates of somatic mutations are expressed in both V H1 of the H chain and in V κ1C of the L chain. The V H36–60 is expressed in antibodies with the major Id of the azophenyl-arsonate (Ars) response and V HOXl generally contributes to the phenyl-oxazolone specificity. This suggests that these V genes are involved in the recognition of the azophenyl moiety of the coupled PC hapten. Thus PC-KLH specific IgE antibodies utilize mutated V Hl and/or V H/V L gene segment combinations which are involved in binding of the azophenyl spacer. These IgE are therefore specific for azophenyl-phosphorylcholine, unlike antibodies normally expressed against the Streptococcus PC determinant in mice. The genetic diversity and the high mutation rates indicate that the specific B cells develop later in the immune response. Thus, they represent newly generated specificities of so-called group II anti-PC antibodies and are not isotype-switch descendants from already existing T15 Id positive IgM antibodies.