Rapid deletion of rearranged T cell antigen receptor (TCR) Vα-Jα segment by secondary rearrangement in the thymus: Role of continuous rearrangement of TCR α chain gene and positive selection in the T cell repertoire formation

Abstract

A rearranged T cell receptor (TCR) V␣ and J␣ gene from a cytochrome c-specific T cell hybridoma was introduced into the genomic J␣ region. The introduced TCR ␣ chain gene is expressed in a majority of CD3 positive and CD4 CD8 double-negative immature thymocytes. However, only a few percent of the double-positive and single-positive thymocytes express this TCR ␣ chain. This decrease is caused by a rearrangement of TCR ␣ chain locus, which deletes the introduced TCR gene. Analysis of the mice carrying the introduced TCR ␣ chain and the transgenic TCR ␤ chain from the original cytochrome c-specific T cell hybridoma revealed that positive selection efficiently rescues double-positive thymocytes from the loss of the introduced TCR ␣ chain gene. In the mice with negatively selecting conditions, T cells expressing the introduced TCR ␣␤ chains were deleted at the double-positive stage. However, a large number of thymocytes escape negative selection by using an endogenous TCR ␣ chain created by secondary rearrangement maintaining normal thymocyte development. These results suggest that secondary rearrangements of the TCR ␣ chain gene play an important role in the formation of the T cell repertoire.A productive VDJ rearrangement of T cell antigen receptor (TCR) ␤ chain inhibits a secondary TCR ␤ gene rearrangement (1), a process called ''allelic exclusion.'' This process ensures that each T cell expresses only one TCR ␤ chain. In contrast, VJ rearrangement of the TCR ␣ chain is not subject to allelic exclusion, and the expression of a functional TCR ␣ chain does not inhibit another TCR ␣ gene rearrangement (2-4). A pre-existing, functionally rearranged V␣-J␣ segment can be deleted by a secondary rearrangement on the same chromosome (5). These results clearly demonstrate that TCR ␣ and ␤ chain genes are regulated differently for the initiation of rearrangement and for allelic exclusion. The mechanisms controlling these differences are not known, and the biological significance of this differential regulation is not understood.To study the regulation of TCR ␣ chain rearrangement and expression during T cell maturation in the thymus, we established a genetically modified mouse line. In this knock-in (KI) mouse, the rearranged TCR V␣J␣ gene from a cytochrome c (Cyt c)-specific T cell hybridoma, 2B4 (6), is inserted in the 5Ј region of the germ-line J␣ locus by using homologous recombination in an embryonic stem (ES) cell. Analysis of the TCR ␣ chain KI mice revealed a rapid deletion of the introduced TCR ␣ gene in the CD4 CD8 double-positive (DP) stage by rearrangement of endogenous TCR ␣ chains. This finding indicates that the TCR ␣ chain locus continuously rearranges and deletes pre-existing functionally rearranged VJ segments in vivo. By crossing the KI mouse to the 2B4 TCR ␤ chain transgenic mouse, we demonstrate that the positive selection of T cells efficiently terminated secondary rearrangements and prevented deletion of the introduced TCR ␣ chain gene. Furthermore, we demonstrate that T cells expressing the KI...

Impact of early expression of TCR α chain on thymocyte development

“…The majority of DP thymocytes in the 2B4 § -KI mice are therefore indistinguishable from wild-type DP thymocytes both genetically and phenotypically [14], and should exhibit normal survival kinetics and DP-to-SP transition efficiency. The observation that the 2B4 § -TG and 2B4 § -KI mice have similarly decreased DP thymocyte numbers thus argues that the phenotype-inducing alteration does occur neither at the DP stage nor during DP-to-SP transition, but instead during the DN-to-DP transition.…”

“…secondary rearrangement of the endogenous § loci and the deletion of the 2B4 § -KI gene [14]. The majority of DP thymocytes in the 2B4 § -KI mice are therefore indistinguishable from wild-type DP thymocytes both genetically and phenotypically [14], and should exhibit normal survival kinetics and DP-to-SP transition efficiency.…”

“…This is achieved because (1) the use of TCR § -but not § gtransgenic mice avoids the potential aberrant § g TCR/ MHC interactions in DN thymocytes that may in itself affect thymocyte development [32]; (2) the decreased DP thymocyte number in D10 § -TG mice potentiates the generalization of this phenomenon to other TCR § -transgenic mice; and (3) the inclusion of the 2B4 § -KI mice in the analyses helps to define the DN-to-DP transition as the major stage of alteration [14]. Our results thus extend beyond previous findings and provide clear in vivo evidences for the detrimental effects of TCR § chain expression on DN-to-DP thymocyte maturation.…”

“…This segregation may in turn allow the rearrangement of the § and g loci to be regulated differently, so that multiple TCR § rearrangements can occur without disturbing the TCR g locus. As existing results suggest that multiple TCR § rearrangements are important for efficient generation of a functional peripheral repertoire [14,43,44], individuals with better segregation of TCR § and g rearrangements and thus better multiple TCR § rearrangements will then pose an advantage during natural selection, which will eventually result in a population in which the majority of the TCR § rearrangements are confined at the DP stage.…”

“…2B4 § -TG and 2B4 § -KI mice express the same TCR § chain with different regulatory elements [13,14], while 2B4 § -TG and D10 § -TG mice express different TCR § chains but use the same TCR transgenic expression vector [13,15]. All three mice express the transgenic § chain prematurely at the DN stage [13][14][15]. Analysis of their thymic cellularity and CD4/CD8 subset distribution showed that the total thymocyte numbers of the 2B4 § -KI, 2B4 § -TG, and D10 § -TG mice were less than˚20% that of B6 mice ( Fig.…”