Revision of T cell receptor α chain genes is required for normal T lymphocyte development

Abstract

To become mature ␣␤ T cells, developing thymocytes must first assemble a T cell receptor (TCR) ␤ chain gene encoding a TCR␤ chain that forms a pre-TCR. These cells then need to generate a TCR␣ chain gene encoding a TCR␣ chain, which, when paired with the TCR␤ chain, forms a selectable ␣␤ TCR. Newly generated VJ␣ rearrangements that do not encode TCR␣ chains capable of forming selectable ␣␤ TCRs can be excised from the chromosome and replaced with new VJ␣ rearrangements. Such replacement occurs through the process of TCR␣ chain gene revision whereby a V␣ gene segment upstream of the VJ␣ rearrangement is appended to a downstream J␣ gene segment. A multistep, gene-targeting approach was used to generate a modified TCR␣ locus (TCR␣ sJ ) with a limited capacity to undergo revision of TCR␣ chain genes. Thymocytes from mice homozygous for the TCR␣ sJ allele are defective in their ability to generate an ␣␤ TCR. Furthermore, those thymocytes that do generate an ␣␤ TCR have a diminished capacity to be positively selected, and TCR␣ sJ/sJ mice have significantly reduced numbers of mature ␣␤ T cells. Together, these findings demonstrate that normal T cell development relies on the ability of developing thymocytes to revise their TCR␣ chain genes.T he checkpoints imposed on developing T cells mandate that they generate T cell receptor (TCR) ␣ and ␤ chains that form an ␣␤ TCR capable of positive selection (1). TCR ␣ and ␤ chain gene assembly is precisely regulated within the context of normal thymocyte development. TCR␤ chain genes are assembled in CD4 Ϫ ͞CD8 Ϫ [double negative (DN)] thymocytes (2-4). Expression of a TCR␤ chain, as a pre-TCR, results in signals that promote cellular expansion and developmental stage progression to the CD4 ϩ ͞CD8 ϩ [double positive (DP)] stage of thymocyte development (5). Pre-TCR signals also promote TCR␣ chain gene assembly and prohibit further TCR␤ chain gene assembly through the process of allelic exclusion (5-7). Because allelic exclusion prohibits DP thymocytes from generating new TCR␤ chains, these cells must generate TCR␣ chains that form an ␣␤ TCR capable of positive selection if they are to become mature T cells.The genes encoding the TCR␣ chain are intermingled with TCR␦ chain genes in the TCR␣͞␦ locus ( Fig. 1) (8). TCR␣ variable region genes are assembled from V␣ and J␣ gene segments. There are Ϸ100 V␣ gene segments clustered in the 5Ј region of the mouse TCR␣͞␦ locus and 61 J␣ gene segments, spanning Ϸ60 kb, clustered in the 3Ј region of the locus (Fig. 1) (8). The V␣ and J␣ gene segments are oriented such that any rearrangement occurs by deletion, excising the intervening DNA from the chromosome (8).Thymocytes entering the DP compartment from the DN compartment express low TCR␤ chain levels, as the pre-TCR, and are referred to as TCR␤ low DP thymocytes. Initial V␣ rearrangements in these cells are targeted to the most 5Ј J␣ gene segments through the activity of the T early ␣ (TEA) promoter (9). A nonproductive VJ␣ rearrangement could be followed by a VJ␣ rearrangement on the oth...

Peripheral T Cell Lymphopenia and Concomitant Enrichment in Naturally Arising Regulatory T Cells: The Case of the Pre-Tα Gene-Deleted Mouse

“…Some i.c.TCR␤ ϩ DP cells may remain CD5 Ϫ because they either lack correct TCR␣␤ pairing or functional TCR␣ rearrangements. Secondary TCR␣ rearrangements might allow some CD5 Ϫ i.c.TCR␤ ϩ DP cells to up-regulate CD5 expression (40). However, our results suggest that only ϳ15% of pT␣ Ϫ/Ϫ DP, namely, i.c.TCR␤ ϩ CD5 ϩ cells, are potentially capable of being positively selected.…”

ATM deficiency impairs thymocyte maturation because of defective resolution of T cell receptor α locus coding end breaks

“…A transgenic Rag-reconstitution model allowed analysis of DP thymocytes that rapidly lose expression of Rag-1 and can only undergo an early, single round of TCR␣ recombination (22). In another model, a modified TCR␣ locus was generated by gene targeting that has a very limited capacity for revision of TCR␣ rearrangement (TCR␣ sJ ) (23). In both of these models, the limited ability to rearrange the TCR␣ locus results in decreased SP maturation, as well as accumulation of the TCR lo DP thymocytes.…”

Dynamic regulation of c‐Myc proto‐oncogene expression during lymphocyte development revealed by a GFP‐c‐Myc knock‐in mouse

“…Generation of c-Myc G/G mice Embryonic stem cell transfection, selection and Cre-mediated deletion were carried out as previously described [28]. The pLNTK-c-Myc-GFP targeting vector was generated using a 4.1-kb SphI-DraIII 5 0 homology arm and a 4.4-kb DraIII-HindIII 3 0 homology arm (Fig.…”

“…Southern and Northern blot analyses were carried out as previously described [28]. Probe A is a 1.7-kb XbaI genomic fragment from the c-Myc locus and probe C is a 0.4-kb HindIIIKpnI fragment from the c-Myc locus.…”