Vβ Recombination Signal Sequences Mediate Monoallelic And Monogenic Tcrβ Gene Assembly: Implications For The Tcrβ Repertoire And Allelic Exclusion In Health And Disease
T Cell Receptor
Allergy and Immunology
Immunology and Infectious Disease
Monoallelic expression of antigen receptor (AgR) genes is assumed to be critical for the proper development and function of T and B lymphocytes. AgR loci are composed of variable (V), joining (J), and sometimes diversity (D) gene segments that must be cleaved and assembled by RAG-endonuclease mediated V(D)J recombination to form a functional gene. TCRβ, IgH, and Igκ allelic exclusion is achieved by: assembly of an in-frame gene on one allele, transient inhibition of V(D)J recombination signaled by RAG DNA double stranded breaks (DSBs), and the resulting protein sending feedback signals to permanently inhibit V-to-(D)J recombination of the non-functional allele. Any definitive mechanisms that promote asynchronous AgR gene assembly in developing lymphocytes remain unknown. V segment recombination signal sequences (RSSs) of Tcrb and Igh loci that target RAG have been proposed to render V recombination inefficient and mediate monoallelic V-to-DJ recombination. To test the role of Vβ RSSs in promoting Tcrb allelic exclusion, I created and studied mice harboring particular Vβ RSS replacements with the stronger 3’Dβ1 RSS. I demonstrate a substantial role for weak Vβ RSSs in limiting: Vβ recombination frequency, biallelic Vβ-to-DJβ recombination before the onset of transient and permanent feedback mechanisms, biallelic TCRβ expression, and unexpectedly dual TCRβ chain expression from a single Tcrb allele. These data indicate that weak Vβ RSSs limit Vβ recombination to promote monogenic Tcrb assembly within the time window before feedback inhibition halts Vβ rearrangements. I also establish a role for ATM, a key factor in the DNA damage response that promotes DNA repair and mediates transient feedback inhibition, in guarding against the formation of nonfunctional deletions for V segments that rearrange by inversion and cooperating with weak Vβ RSSs to impose TCRβ allelic exclusion. AgR allelic exclusion is most stringently applied to Tcrb and Igh, whose assembly, expression, and signaling through pre-AgR complexes drive cellular proliferation. As I am able to drive biallelic recombination of Tcrb genes, I introduce preliminary data that supports the model that mechanisms directing monoallelic induction of RAG DSBs evolved at Tcrb and Igh loci in part to suppress DSBs from entering S phase and forming oncogenic AgR translocations. Finally, as the Vβ RSS replacements result in dramatic shifts in the Vβ repertoire, I present data that suggests the composition of the TCRβ repertoire may affect antigen-specific immune responses by defining the frequency of naïve antigen-reactive cells. Together, these studies reveal mechanisms in developing thymocytes that promote: monoallelic Tcrb assembly and allelic exclusion, repertoire diversity, and perhaps T cell immune responses and genome stability.