The Cyclin D3 Protein Enforces Monogenic TCRß Expression by Mediating TCRß Protein-Signaled Feedback Inhibition of Vß Recombination.

In jawed vertebrates, adaptive immunity depends on the process of V(D)J recombination creating vast numbers of T and B lymphocytes that each expresses unique Ag receptors of uniform specificity. The asynchronous initiation of V-to-(D)J rearrangement between alleles and the resulting protein from one allele signaling feedback inhibition of V recombination on the other allele ensures homogeneous receptor specificity of individual cells. Upon productive Vß-to-DßJß rearrangements in noncycling double-negative thymocytes, TCRß protein signals induction of the cyclin D3 protein to accelerate cell cycle entry, thereby driving proliferative expansion of developing αß T cells. Through undetermined mechanisms, the inactivation of cyclin D3 in mice causes an increased frequency of αß T cells that express TCRß proteins from both alleles, producing lymphocytes of heterogeneous specificities. To determine how cyclin D3 enforces monogenic TCRß expression, we used our mouse lines with enhanced rearrangement of specific Vß segments due to replacement of their poor-quality recombination signal sequence (RSS) DNA elements with a better RSS. We show that cyclin D3 inactivation in these mice elevates the frequencies of αß T cells that display proteins from RSS-augmented Vß segments on both alleles. By assaying mature αß T cells, we find that cyclin D3 deficiency increases the levels of Vß rearrangements that occur within developing thymocytes. Our data demonstrate that a component of the cell cycle machinery mediates TCRß protein-signaled feedback inhibition in thymocytes to achieve monogenic TCRß expression and resulting uniform specificity of individual αß T cells.

Monogenic TCRß Assembly and Expression Are Paramount for Uniform Antigen Receptor Specificity of Individual αß T Lymphocytes.

The ability of individual T and B cells to display Ag receptors of unique uniform specificity is the molecular basis of adaptive immunity. Most αß T cells achieve uniform specificity by assembling in-frame genes on only one allelic copy of TCRß and TCRα loci, while others prevent incorporation of TCRα protein from both alleles into TCRs. Analysis of mice expressing TCR proteins from a restricted combination of transgenes showed that TCR protein pairing restrictions achieve uniform specificity of cells expressing two types of TCRß protein. However, whether this mechanism operates in the physiological context where each dual-TCRß cell expresses one set of a vast number of different TCRß proteins remains an open question, largely because there is a low, but significant, portion of cells carrying two in-frame TCRß genes. To resolve this issue, we inactivated one allelic copy of the TCRα locus in a new mouse strain that assembles two in-frame TCRß genes in an elevated fraction of cells. This genetic manipulation has no effect on the frequency of cells that display multiple types of αß TCR, yet increases the representation of cells displaying TCRß proteins that generate more highly expressed TCRs. Our data demonstrate that some TCRß proteins exhibit differential functional pairing with TCRα proteins, but these restrictions have negligible contribution for ensuring uniform specificity of cells that express two types of TCRß protein. Therefore, we conclude that mechanisms governing monogenic assembly and expression of TCRß genes in individual cells are paramount for uniform specificity of αß T lymphocytes.

Poor-Quality Vß Recombination Signal Sequences and the DNA Damage Response ATM Kinase Collaborate to Establish TCRß Gene Repertoire and Allelic Exclusion.

The monoallelic expression (allelic exclusion) of diverse lymphocyte Ag receptor genes enables specific immune responses. Allelic exclusion is achieved by asynchronous initiation of V(D)J recombination between alleles and protein encoded by successful rearrangement on the first allele signaling permanent inhibition of V rearrangement on the other allele. The ATM kinase that guides DNA repair and transiently suppresses V(D)J recombination also helps impose allelic exclusion through undetermined mechanisms. At the TCRß locus, one Vß gene segment (V31) rearranges only by inversion, whereas all other Vß segments rearrange by deletion except for rare cases in which they rearrange through inversion following V31 rearrangement. The poor-quality recombination signal sequences (RSSs) of V31 and V2 help establish TCRß gene repertoire and allelic exclusion by stochastically limiting initiation of Vß rearrangements before TCRß protein-signaled permanent silencing of Vß recombination. We show in this study in mice that ATM functions with these RSSs and the weak V1 RSS to shape TCRß gene repertoire by restricting their Vß segments from initiating recombination and hindering aberrant nonfunctional Vß recombination products, especially during inversional V31 rearrangements. We find that ATM collaborates with the V1 and V2 RSSs to help enforce allelic exclusion by facilitating competition between alleles for initiation and functional completion of rearrangements of these Vß segments. Our data demonstrate that the fundamental genetic DNA elements that underlie inefficient Vß recombination cooperate with ATM-mediated rapid DNA damage responses to help establish diversity and allelic exclusion of TCRß genes.

The RAG1 Ubiquitin Ligase Domain Stimulates Recombination of TCRß and TCRα Genes and Influences Development of αß T Cell Lineages.

RAG1/RAG2 (RAG) endonuclease-mediated assembly of diverse lymphocyte Ag receptor genes by V(D)J recombination is critical for the development and immune function of T and B cells. The RAG1 protein contains a ubiquitin ligase domain that stabilizes RAG1 and stimulates RAG endonuclease activity in vitro. We report in this study that mice with a mutation that inactivates the Rag1 ubiquitin ligase in vitro exhibit decreased rearrangements and altered repertoires of TCRß and TCRα genes in thymocytes and impaired thymocyte developmental transitions that require the assembly and selection of functional TCRß and/or TCRα genes. These Rag1 mutant mice present diminished positive selection and superantigen-mediated negative selection of conventional αß T cells, decreased genesis of invariant NK T lineage αß T cells, and mature CD4+ αß T cells with elevated autoimmune potential. Our findings reveal that the Rag1 ubiquitin ligase domain functions in vivo to stimulate TCRß and TCRα gene recombination and influence differentiation of αß T lineage cells, thereby establishing replete diversity of αß TCRs and populations of αß T cells while restraining generation of potentially autoreactive conventional αß T cells.