Vβ recombination signal sequences enforce TCRβ allelic exclusion

Abstract

Abstract The assembly of antigen receptor (AgR) genes via RAG1/RAG2 (RAG) mediated recombination of variable (V), diversity (D), and joining (J) gene segments is essential for adaptive immunity. V(D)J recombination of Tcrb, Igh, and Igk loci is regulated such that functional AgR genes are assembled on and expressed from one allele (allelic exclusion) in most cells. It is assumed that allelic exclusion is achieved by mono-allelic initiation and feedback inhibition of the V-to-(D)J recombination step, and intrinsic features of recombination signal sequences (RSSs) have been proposed to mediate mono-allelic V-to-DJ recombination of Tcrb and Igh loci. To investigate the role of Vβ RSSs in enforcing allelic exclusion, we established and analyzed mice with the Trbv2 or Trbv31 RSS replaced by a RSS possessing greater recombination activity. Each of these RSS substitutions causes a profound increase in the development of T cells expressing either Trbv2+ or Trbv31+ TCRβ chains at the expense of cells expressing other TCRβ chains, reflecting that the RSSs flanking Vβ segments are major determinants of Vβ recombination frequency. Each RSS replacement also promotes a modest increase in the fraction of cells expressing Trbv2+ or Trbv31+ TCRβ chains along with another type of TCRβ chain. Strikingly, mice with Trbv2 and Trbv31 RSS replacements on opposite alleles have a 30-fold higher than normal level of bi-allelic Trbv2+ and Trbv31+ expression, demonstrating that the RSSs flanking Vβ segments enforce TCRβ allelic exclusion. Our data suggests that the infrequency of Vb recombination, as determined by weak Vβ RSSs, limits bi-allelic assembly of Tcrb genes within the time window before feedback inhibition permanently cements allelic exclusion.