New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling

Abstract

To gain more insight into initiation and regulation of T cell receptor (TCR) gene rearrangement during human T cell development, we analyzed TCR gene rearrangements by quantitative PCR analysis in nine consecutive T cell developmental stages, including CD34+ lin- cord blood cells as a reference. The same stages were used for gene expression profiling using DNA microarrays. We show that TCR loci rearrange in a highly ordered way (TCRD-TCRG-TCRB-TCRA) and that the initiating Ddelta2-Ddelta3 rearrangement occurs at the most immature CD34+CD38-CD1a- stage. TCRB rearrangement starts at the CD34+CD38+CD1a- stage and complete in-frame TCRB rearrangements were first detected in the immature single positive stage. TCRB rearrangement data together with the PTCRA (pTalpha) expression pattern show that human TCRbeta-selection occurs at the CD34+CD38+CD1a+ stage. By combining the TCR rearrangement data with gene expression data, we identified candidate factors for the initiation/regulation of TCR recombination. Our data demonstrate that a number of key events occur earlier than assumed previously; therefore, human T cell development is much more similar to murine T cell development than reported before.

Figure 1.

RQ-PCR analysis of TCRD and TCRG gene rearrangements in human T cell development and mature T cells. (A) Schematic diagram of the human TCRD gene complex. The six “classical” Vδ gene segments (blue) are scattered between Vα gene segments (black). Because Vδ4, Vδ5, and Vδ6 also are recognized as Vα gene segments, their Vα gene code is given in parentheses (adapted from reference 9). (B) Analysis of Dδ2-Dδ3 rearrangement. (C) Analysis of Dδ2-Jδ1 rearrangement. (D) Analysis of Vδ1-Jδ1 and Vδ2-Jδ1 rearrangements. (E) Analysis of Vδ-Dδ3 rearrangement. (F) Schematic diagram of the human TCRG gene complex. Only the rearrangeable Vγ gene segments are depicted in blue (functional Vγ) or gray (nonfunctional Vγ). For the Jγ gene segments both nomenclatures are used (adapted from reference 9). (G) Analysis of Vγ to Jγ1.1 and Jγ2.1 rearrangements. (H) Analysis of Vγ to Jγ1.3 and Jγ2.3 rearrangements. (I) Analysis of Vγ to Jγ1.2 rearrangements.

Figure 2.

RQ-PCR and GeneScan analysis of TCRB rearrangements in human T cell development and in mature T cells. (A) Schematic diagram of the human TCRB gene complex. (B) Analysis of Dβ to Jβ rearrangements. (C) Schematic diagram of Vβ-Jβ rearrangements as determined by GeneScan analysis. The primers for the Jβ1 cluster were hexachloro-6-carboxy-fluorescein (HEX)-labeled, whereas primers for the Jβ2 cluster were 6-carboxy-fluorescein (FAM)-labeled and yielded a green or blue signal upon GeneScan analysis, respectively. Analysis of Vβ-Jβ rearrangements in CD34⁺CD38⁺CD1a⁺ cells (d), ISP cells (e), DP CD3⁻ cells (f), TCRαβ⁺ thymocytes (g) and TCRγδ⁺ thymocytes (h).

Figure 3.

RQ-PCR analysis of initiating events around TCRA rearrangement in human T cell development and mature T cells. (A) Schematic diagram of the human TCRA gene complex. The TEA element (red) forms a sterile mRNA with Cα sequences. The TCRD deleting elements δRec and ψJα are also indicated. (B) Analysis of TEA-Cα mRNA expression. Due to shortage of material, TEA expression was only determined once. (C) Analysis of the TCRD deleting rearrangement δRec-ψJα. (D) Analysis of “remaining” TCRA germline DNA.

Figure 4.

Analysis of probe sets related to transcriptional regulation. (A) Hierarchical clustering of 446 probe sets differentially expressed over the various stages and related to transcriptional regulation and DNA binding. (B) z-score trend representation for each cluster deduced from the 446 probe sets. Cluster codes (–15) are shown in the top right corner, z-score on the y axis, subsets on the x axis. The gray area represents 1 SD. The asterisk in the top right corner indicates clusters that contain genes that are potentially important in regulating TCR rearrangements. N, indicates the number of probe sets in a cluster.