Restriction of endogenous T cell antigen receptor β rearrangements to Vβ14 through selective recombination signal sequence modifications

Abstract

In CD4Ϫ /CD8 Ϫ (double-negative, DN) thymocytes, TCR␤ gene segments are assembled in an ordered manner with D␤ to J␤ rearrangements occurring on both alleles before joining of V␤ segments to a preexisting DJ␤ complex (7). In-frame (productive) V␤DJ␤ rearrangements generate TCR␤ chains. At this stage, pairing of TCR␤ chains with the preexisting pT␣ chain to form preTCRs generates signals that lead to expansion of DN cells and their further development into CD4 ϩ / CD8 ϩ (double-positive, DP) thymocytes and the rearrangement of TCR␣ genes (8,9). If the first TCR␤ allele that undergoes V␤ to DJ␤ rearrangement generates a productive TCR␤ chain that pairs with pT␣, V␤ to DJ␤ rearrangement on the second allele is thought to be inhibited by a feedback mechanism that may serve to help enforce TCR␤ allelic exclusion (8, 9). However, out-of-frame V␤DJ␤ rearrangements, or rearrangements that do not lead to a TCR␤ chain that pairs with the preT␣ chain, on the first allele permit V␤ to DJ␤ rearrangements on the second allele that, if productive, generate a TCR␤ chain that pairs with pT␣ to signal differentiation. In DP thymocytes, productive assembly of TCR␣ variable region exons can lead to the generation of TCR␣ chains that, if they pair with TCR␤ chains, form ␣␤ TCRs and signal differentiation to the CD4 ϩ /CD8 Ϫ or CD4 Ϫ /CD8 ϩ (single-positive, SP) thymocyte stage (8, 9). Developmental stage-specific rearrangement of TCR␤ and TCR␣ genes and TCR␤ allelic exclusion are thought to be mediated, at least in part, through modulation of V(D)J recombinational accessibility of participating gene segments to the RAG endonuclease (8, 9). In the TCR␤ locus, V␤s have a 23-RS, J␤s a 12-RS, and D␤s a 5Ј 12-RS and a 3Ј 23-RS. Despite apparent 12/23 compatibility of V␤ 23-RSs and J␤1 12-RSs, the 5ЈD␤1 12-RS, but not the J␤1 12-RSs, can target endogenous V␤ rearrangements (10). This ''beyond 12/23 (B12/23) restriction'' can be recapitulated in transfected plasmid substrates and occurs at the level of RAG cutting of DNA substrates in vitro (11,12). The latter result demonstrates that functional synapses can occur between the RAG proteins and V␤/D␤ RSs but not V␤/J␤ RSs. In contrast,

TCRβ Feedback Signals Inhibit the Coupling of Recombinationally Accessible Vβ14 Segments with DJβ Complexes

“…Of the 67 Vβ14 Rep/WT αβ T cell hybridomas analyzed, 47 (73%) contained Vβ14 Rep recombination events (Table II). This observation is consistent with our earlier findings obtained from chimeric mice that Vβ14 is RAG accessible in a substantially higher percentage of thymocytes than the approximately 7% in which Vβ14-to-DJβ rearrangements occur (17, 19, 64). Unexpectedly, 52 of the 66 (79%) Vβ8 Tg :Vβ14 Rep/WT αβ T cell hybridomas and 105 of the 185 (58%) Vβ14 Rep/NT αβ T cell hybridomas contained Vβ14 Rep recombination events (Table II).…”

TCRβ Allelic Exclusion in Dynamical Models of V(D)J Recombination Based on Allele Independence

“…These estimates are in accordance with assessments on TCRb allelically included cells based on genomic sequencing of a number of VbDJb joints (3-10%; even though, at the phenotypic level, dual TCRb + cells represent a smaller percentage due to postrearrangement events, such as competition between Vb gene promoters, TCR b-and a-chain pairing, etc.) (24,25,44,45). In addition, in the case of the b LD/LD mice, the predicted proportions of sVDJ + / VDJ + cells are similar to those in WT animals and thus corroborate experimental observations arguing that the specific mutation does not impact on TCRb allelic exclusion (23).…”

Productive Coupling of Accessible Vβ14 Segments and DJβ Complexes Determines the Frequency of Vβ14 Rearrangement

“…In contrast, transgenic overexpression of TCR␤ chains may inhibit endogenous rearrangements, at least to some degree, by accelerated development and not normal feedback mechanisms (41). In this context, sequence analyses of limited numbers of V␤DJ␤ joins in WT ␣␤ T cells and direct V␤14 to J␤ rearrangements in ␣␤ T cells with specific TCR␤ RS replacements revealed two in-frame rearrangements in 5-10% of cells (33,42,43), indicating that normal TCR␤ expression may not inhibit V␤ to DJ␤ rearrangement and lead to allelic exclusion in all developing ␣␤ T cells. Accordingly, our observation that V␤14…”

“…Rep recombinational accessibility largely mirrors that of endogenous V␤14 rearrangements, which are readily detectable in DNIII, but not DNII, thymocytes (33).…”