The large repertoire of Ig and TCR derive from combinatorial diversity of many V, D and J segments as well as from diversity at the junctions of those segments. Junctional diversity is derived both from the deletion of nucleotides from the coding regions and from the addition of non-templated N region nucleotides before ligation of the segments into a contiguous gene. N region nucleotides have been shown to be added by the enzyme terminal deoxytransferase (TdT). We have shown that Ig and alphabeta TCR from fetal and newborn mice have few N regions and overall there is much less junctional diversity than in adults. Thus, the neonatal Ig and TCR repertoires are very much smaller than, and different from, the adult repertoires. To address the question of the importance of this two-tiered immune repertoire we will create mice which have only the neonatal repertoire of N- receptors (TdT-), or only the adult N+ repertoire (TdT+). The TdT- mouse will be constructed by gene targeting via homologous recombination in ES cells. The N+ mouse will be a transgenic mouse with a TdT transgene under the control of the IgH enhancer. We have preliminary data suggesting that the lack of N regions may hinder the T cell's progress through the selective processes in the thymus. Therefore, we may observe changes in the numbers or composition of mature thymocytes or peripheral T cells in the TdT-altered mice. Likewise, many peripheral B cells (both conventional and CD5+) have already undergone ligand-induced expansion, and therefore the mice with altered immune repertoires may have a different composition of peripheral B cells than normal animals. Another hypothesis which can be addressed with these mice is our proposal that the non-random Vh usage in fetal life may be due to targeted recombination via short sequence homologies at the ends of the coding regions. Such homologies would be predicted to preferentially make in-frame V-D-J rearrangements with the Vh gene families which are overrepresented in the neonate, and out-of-frame rearrangements with the Vh families which are underrepresented early in life. TdT- mice would be predicted to maintain the fetal Vh family distribution. The TdT- mice will be analyzed for any manifestations of autoimmune disease. the B and T cell repertoires will be probed by immunizing with a variety of immunogens, to determine if there are significant differences or """"""""holes"""""""" in the repertoire. Finally, although it has been conclusively shown that TdT can add non-templated nucleotides to junctions, it is not known if any other enzymes or mechanisms exist to create non-templated junctional diversity. Deletion of TdT will answer that issue.
| Nadel, B; Tehranchi, S; Feeney, A J (1995) Coding end processing is similar throughout ontogeny. J Immunol 154:6430-6 |
| Bain, G; Maandag, E C; Izon, D J et al. (1994) E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements. Cell 79:885-92 |
