TCR beta feedback signals inhibit the coupling of recombinationally accessible V beta 14 segments with DJ beta complexes

Abstract

Ag receptor allelic exclusion is thought to occur through monoallelic initiation and subsequent feedback inhibition of recombinational accessibility. However, our previous analysis of mice containing a V(D)J recombination reporter inserted into Vbeta14 (Vbeta14(Rep)) indicated that Vbeta14 chromatin accessibility is biallelic. To determine whether Vbeta14 recombinational accessibility is subject to feedback inhibition, we analyzed TCRbeta rearrangements in Vbeta14(Rep) mice containing a preassembled in-frame transgenic Vbeta8.2Dbeta1Jbeta1.1 or an endogenous Vbeta14Dbeta1Jbeta1.4 rearrangement on the homologous chromosome. Expression of either preassembled VbetaDJbetaC beta-chain accelerated thymocyte development because of enhanced cellular selection, demonstrating that the rate-limiting step in early alphabeta T cell development is the assembly of an in-frame VbetaDJbeta rearrangement. Expression of these preassembled VbetaDJbeta rearrangements inhibited endogenous Vbeta14-to-DJbeta rearrangements as expected. However, in contrast to results predicted by the accepted model of TCRbeta feedback inhibition, we found that expression of these preassembled TCR beta-chains did not downregulate recombinational accessibility of Vbeta14 chromatin. Our findings suggest that TCRbeta-mediated feedback inhibition of Vbeta14 rearrangements depends on inherent properties of Vbeta14, Dbeta, and Jbeta recombination signal sequences.

Figure 1. Vβ8^Tg and Vβ14^NT/WT mice exhibit TCRβ allelic exclusion

(A) Schematic representation of the transgenic Vβ8^Tg and endogenous Vβ14^NT loci. Open boxes depict Vβ, Dβ, Jβ, and Cβ segments. Closed circles depict the TCRβ enhancer (Eβ) and the promoters of the pre-assembled VβDβJβ rearrangements, with arrows indicating the direction of sense VβDβJβ transcripts. Triangles show the 5'Dβ1/Vβ14 RSS join. (B) Shown are representative anti-Cβ, by anti-Vβ8 or anti-Vβ14 FACS analysis of cells isolated from the thymuses and spleens of wild-type, Vβ8^Tg, and Vβ14^NT/WT mice.

Figure 2. Grossly normal αβ T cell development in Vβ8^Tg and Vβ14^NT/WT mice

(A) Bar graphs showing the average number of thymocytes and splenocytes from at least five mice of each genotype. The error bars are standard error of the mean. (B) Shown are representative anti-CD4 and anti-CD8 FACS analysis of cells isolated from the thymuses and spleens of wild-type, Vβ8^Tg, and Vβ14^NT/WT mice. The percentage of DN, DP, CD4⁺ SP, and CD8⁺ SP thymocytes and CD4⁺ and CD8⁺ αβ T cells is depicted. (C–D) Bar graphs showing the average frequency of (C) DN, DP, CD4⁺ SP, and CD8⁺ SP thymocytes and (D) CD4⁺ and CD8⁺ αβ T cells from at least five mice of each genotype. The error bars are standard error of the mean. Significant differences have been calculated using a two-tailed Student's t-test.

Figure 3. Accelerated early thymocyte development in Vβ8^Tg and Vβ14^NT/WT mice

(A) Shown are representative anti-CD117 and anti-CD25 FACS analysis of thymocytes gated on cells negative for mature cells markers (TCRβ, TCRδ, CD4, CD8α, CD19, CD11c, CD11b, B220 and NK1.1). (B) Bar graphs showing the average frequency of ETP, DNII, DNIII, DNIII/DNIV, and DNIV thymocytes at least five mice of each genotype. The error bars are standard error of the mean. Significant differences have been calculated using a two-tailed Student's t-test.

Figure 4. β-selection is enhanced in Vβ8^Tg and Vβ14^NT/WT mice

(A) Shown are representative histograms of anti-Vβ8 and anti-Vβ14 FACS analysis of DNIII thymocytes isolated from wild-type, Vβ8^Tg, and Vβ14^NT/WT mice. (B) Shown are representative Western blot analysis of cyclin D3 and tubulin protein expression in total thymocytes of wild-type, Vβ8^Tg, and Vβ14^NT/WT mice. (C) Shown are representative BrdU FACS analysis of ETP, DNII, DNIII, and DNIV thymocytes of wild-type, Vβ8^Tg, and Vβ14^NT/WT mice. The percentage of BrdU⁺ cells is indicated. (D) Shown are representative anti-Cδ and anti-Cβ FACS analysis of cells isolated from the thymuses and spleens of wild-type, Vβ8^Tg, and Vβ14^NT/WT mice. The percentage of Cδ⁺ T cells is depicted.

Figure 5. Vβ14 recombinational accessibility is maintained in thymocytes expressing a pre-assembled TCRβ gene

(A) Schematic representation of germline and DJβ rearranged Vβ14^Rep alleles indicating the positions of the PCR primers and probe used analysis of Vβ14^Rep Dβ-to-Jβ recombination events. The sizes of PCR products amplified from germline and DJβ rearranged Vβ14^Rep alleles are indicated. (B) PCR analysis of Vβ14^Rep Dβ-to-Jβ recombination events conducted on equal amounts of genomic DNA isolated from sort-purified DN and DP thymocytes of Vβ14^Rep/WT and Vβ14^Rep/NT mice. Shown are short and long exposures with the PCR product identities indicated. (C) Schematic representations of germline wild-type TCRβ (Vβ14^WT) and Vβ14^Rep loci, as well as the configurations of Vβ14^Rep alleles that have undergone Vβ14^Rep Dβ-to-Jβ rearrangements or the rearrangement of endogenous (End) Vβ, Dβ, or Jβ segments to the Vβ14^Rep Dβ1 segment. Open boxes depict Vβ, Dβ, Jβ, and Cβ segments. Black triangles indicate 23-RSs and open triangles indicate 12-RSs. Also labeled are the signal join (SJ) formed during endogenous Jβ rearrangements to Vβ14^Rep, the Dβ1-Dβ1 coding join formed during endogenous Dβ rearrangements to the Vβ14^Rep Dβ1 segment, and the Vβ1-Dβ1 coding join formed during endogenous Vβ rearrangements to the Vβ14^Rep Dβ1 segment.