Primary repertoire expansion by junctional diversity in Bos taurus (P1445)

Abstract B cell development varies significantly between species. In many domestic species fetal and neonatal gut-associated lymphoid tissue is essential for the generation of the primary antibody repertoire due to limited range of Ig genes. In fetal ruminants, the repertoire is expanded by somatic hypermutation (SHM) driven by activation-induced cytidine deaminase in the absence of external antigens. In addition to SHM also other mechanisms are likely to contribute to the repertoire expansion. Junctional diversity (JD) created during the recombination process might play a substantial role in bovine fetal IgH. To further characterize the significance of JD, we studied the tissue-specific expression of terminal deoxynucleotidyl transferase (TdT) using RT-qPCR and immunostaining. We also constructed and sequenced several IgH cDNA libraries of different tissues and analysed the junction formation between V(D)J sequences. Our work demonstrates that in fetal bovine B lymphocytes TdT is primarily expressed in the bone marrow. B lymphocytes in other lymphoid tissues did not show elevated TdT expression levels. V(D)J junctions were heterogeneous in sequence, suggesting the addition of N-nucleotides by TdT. In addition, spectratyping revealed significant differences in the distribution of CDR3 lengths between tissues. Our results suggest that along with SHM, TdT-mediated JD contributes significantly to the bovine primary antibody repertoire in fetal life.