Kinetics of T cell receptor beta, gamma, and delta rearrangements during adult thymic development: T cell receptor rearrangements are present in CD44(+)CD25(+) Pro-T thymocytes

Abstract

We performed a comprehensive analysis of T cell receptor (TCR) gamma rearrangements in T cell precursors of the mouse adult thymus. Using a sensitive quantitative PCR method, we show that TCRgamma rearrangements are present in CD44(+)CD25(+) Pro-T thymocytes much earlier than expected. TCRgamma rearrangements increase significantly from the Pro-T to the CD44(-)CD25(+) Pre-T cell transition, and follow different patterns depending on each Vgamma gene segment, suggesting that ordered waves of TCRgamma rearrangement exist in the adult mouse thymus as has been described in the fetal mouse thymus. Recombinations of TCRgamma genes occur concurrently with TCRdelta and D-Jbeta rearrangements, but before Vbeta gene assembly. Productive TCRgamma rearrangements do not increase significantly before the Pre-T cell stage and are depleted in CD4(+)CD8(+) double-positive cells from normal mice. In contrast, double-positive thymocytes from TCRdelta-/- mice display random proportions of TCRgamma rearranged alleles, supporting a role for functional TCRgamma/delta rearrangements in the gammadelta divergence process.

Figure 1

Analysis of Vγ1.2-Jγ2 rearrangements in early thymic progenitors. (A) Schematic representation of the TCRγ locus and of the Vγ1.2-Jγ2 PCR assay. The name of the Vγ, Jγ segments according to the two different nomenclatures are indicated, as is the position of the oligonucleotide primers utilized for the Vγ1.2-Jγ2 amplification reaction. (B) Sort scheme used for obtaining the four CD44⁺CD25⁺ Pro T, CD44^+/−CD25⁺ post-Pre, CD44⁻CD25⁺ Pre-T^, and CD44⁻CD25⁻ post-Pre-T thymocyte subsets. The latter were purified from TN thymocytes after staining with anti-CD44 and anti-CD25 antibodies, as described in Materials and Methods. Sort purities were at least 99.9% upon reanalysis (>99.95% for CD44⁺CD25⁺ thymocytes). The corresponding Pro-T, post-Pro-T, Pre-T, and post-Pre-T nomenclature used in the text is shown. (C) PCR analyses of Vγ1.2-Jγ2 rearrangements were performed for the four populations indicated in B. The amount of amplified Thy-1 product was representative of the quantity of input DNA.

Figure 2

Sequential rearrangements of TCRγ (A) and TCRδ (B) genes during early thymic development. The various V-J PCR assays were performed using different aliquots of the same DNA suspension prepared from each of the four early thymic populations described, as well as for DP cells for TCRδ rearrangements. Southern blots of the products were probed with labeled primer probes specific to each Vγ/δ gene segment.

Figure 3

Analysis of TCR D-Jβ rearrangements during early thymic development. (A) Schematic representation of the PCR assay. The position of the primers as well as the different fragments amplified are shown. (B) Represents a long exposure of the blot obtained as described in experimental procedures after applying the assay described in A on DNA purified from the different early thymic subsets.