Increase of TCR Vβ Accessibility within Eβ Regulatory Region Influences its Recombination Frequency But Not Allelic Exclusion

Abstract

Seventy percent of the murine TCRβ locus (475 kb) was deleted to generate a large deleted TCRβ (βLD) allele to investigate a possible linkage between germline transcription, recombination frequency, and allelic exclusion of the TCR Vβ genes. In these βLD/LD mice, the TCRβ gene locus contained only four Vβ genes at the 5′ side of the locus, and consequently, the Vβ10 gene was located in the original Dβ1-Jβ1cluster within the Eβ regulatory region. We showed that the frequency of recombination and expression of the Vβ genes are strongly biased to Vβ10 in these mutant mice even though the proximity of the other three 5′Vβ genes was also greatly shortened toward the Dβ-Jβ cluster and the Eβ enhancer. Accordingly, the germline transcription of the Vβ10 gene in βLD/LD mice was exceptionally enhanced in immature double negative thymocytes compared with that in wild-type mice. During double negative-to-double positive transition of thymocytes, the level of Vβ10 germline transcription was prominently increased in βLD/LD recombination activating gene 2-deficient mice receiving anti-CD3ε Ab in vivo. Interestingly, however, despite the increased accessibility of the Vβ10 gene in terms of transcription, allelic exclusion of this Vβ gene was strictly maintained in βLD/LD mice. These results provide strong evidence that increase of Vβ accessibility influences frequency but not allelic exclusion of the TCR Vβ rearrangement if the Vβ gene is located in the Eβ regulatory region.

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

“…(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). Overall, the ability of the model to integrate predominant and rare subpopulations and to satisfactorily predict their statistical behaviors is testimony to the power of the approach.…”

“…3A; similar distributions were obtained using parameters from WT data in Ref. 23; E. Farcot and B. Fernandez, unpublished observations). Specifically, the fraction of sVDJ + /VDJ + cells turned out to be fairly low in all three cases, varying monotonically with t f (Fig.…”

“…In this regard, the t VDJ .. t DJ picture accommodates the assumption generally endorsed in the literature that the control of the initiation phase of allelic exclusion (allelic asynchronism) at the TCRb locus impinges on the Vb-to-DJb rearrangement step (1,2,20,42). In addition, the longer GL→DJ and, conversely, slightly shorter DJ→VDJ transitions observed in parallel between b LD/LD mutant and WT T cells represent provocative results as the b LD mutation: 1) specifically enforces the sole usage of the D2-J2 cluster, the segment assembly of which is suspected to proceed less efficiently than that involving the D1-J1 cluster; and 2) greatly reduces the numbers of 59 Vb gene segments and the intervening distance from the D2-J2 cluster, possibly increasing their recombination rate (23,39,43).…”

“…2 (for a practical application of the global approach, also see Materials and Methods, Least-squares fitting procedure and parameter estimation: worked example). Thus, fitting the model to quantitative data from hybridomas generated in two studies using WT mice (22,23) and, in one case, also mice carrying a modified TCRb locus impinging on some aspects of gene recombination (23, 35) yielded parameter triples that aligned onto a smooth curve in all cases ( Fig. 2; each individual triple specifying the model so as to account for the given data set).…”

“…Numbers of b-selected T lymphocytes well-defined in terms of TCRb genotype (Table II) were derived from data sets available in studies by Khor and Sleckman (22) and Senoo et al (23), which used peripheral T cell-derived, cloned hybridomas from WT (22,23) and engineered mutant b LD/LD (23) mice, respectively. Distributed numbers of early-developing double-negative (DN) thymocytes prior to b-selection (Table III) were derived from data sets in studies by Aifantis et al (24,25), which used FACS-sorted intracytoplasmic TCRb + , cell-surface CD25 + small DN cells from WT and pTa-or SPL-76-deficient animals (pTa 2/2 and SPL-76 2/2 , respectively).…”