RECOMBINANT THYROID PEROXIDASE-SPECIFIC AUTOANTIBODIES .1. HOW DIVERSE IS THE POOL OF HEAVY AND LIGHT-CHAINS IN IMMUNOGLOBULIN GENE LIBRARIES CONSTRUCTED FROM THYROID TISSUE-INFILTRATING PLASMA-CELLS

Thyroid peroxidase (TPO) autoantibodies are a distinguishing feature o f autoimmune thyroid disease. We have previously constructed immunoglo bulin G heavy (H) and light (L) chain cDNA libraries from intrathyroid al B-cells. TPO-selected autoantibodies expressed by combined H and L chain libraries (combinatorial libraries) recognized a limited number of epitopes on TPO and used only a few of the many H and L chain varia ble region genes present in the genome (germline genes). One possible explanation for this restriction is a lack of diversity in the parenta l H and L chain gene libraries used to construct the combinatorial lib rary. To address this issue, we determined the nucleotide sequences of randomly selected H and kappa L chain variable region genes from a pa ir of H and L chain libraries. The 12 H chain gene sequences analyzed were highly diverse, and none resembled the genes of TPO-selected auto antibodies. The sequences of 14 randomly selected kappa L chain genes were less diverse; 12 of 14 were closely related to the same germline gene (KL012) used by TPO-specific autoantibodies. However, we observed previously that only about 1 in 500 of the L chains in this library c an pair with an H chain and bind TPO. We now find that, with 1 excepti on, the randomly selected KL012-like genes in the L chain library diff er significantly from the antigen-specific KL012-like genes, particula rly in the antigen-binding regions. In summary, the present data indic ate that 1) the restricted number of H chain genes used by TPO-specifi c autoantibodies cannot be ascribed to limited H chain gene diversity in the parent library; and 2) L chains from combinatorial libraries (e ven when related to the same germline gene) cannot simply be regarded as plastic, or promiscuous, partners for high affinity antigen binding by a particular H chain.