Genetic modulation of T cell receptor gene segment usage during somatic recombination.

Lymphocyte antigen receptors are not encoded by germline genes, but rather are produced by combinatorial joining between clusters of gene segments in somatic cells. Within a given cluster, gene segment usage during recombination is thought to be largely random, with biased representation in mature T lymphocytes resulting from protein-mediated selection of a subset of the total repertoire. Here we show that T cell receptor D beta and J beta gene segment usage is not random, but is patterned at the time of recombination. The hierarchy of gene segment usage is independent of gene segment proximity, but rather is influenced by the ability of the flanking recombination signal sequences (RSS) to bind the recombinase and/or to form a paired synaptic complex. Importantly, the relative frequency of gene segment usage established during recombination is very similar to that found after protein-mediated selection, suggesting that in addition to targeting recombinase activity, the RSS may have evolved to bias the naive repertoire in favor of useful gene products.

Precursor thymocyte proliferation and differentiation are controlled by signals unrelated to the pre-TCR.

In-frame rearrangement of the TCR-beta locus and expression of the pre-TCR are compulsory for the production of CD4+8+ thymocytes from CD4-8- precursors. Signals delivered via the pre-TCR are thought to induce the differentiation process as well as the extensive proliferation that accompanies this transition. However, it is equally possible that pre-TCR expression is required for the success of this transition, but does not play a direct role in the inductive process. In the present manuscript we examine this possibility using a variety of normal and genetically modified mouse models. Our evidence shows that differentiation and mitogenesis can both occur independently of pre-TCR expression. However, these processes are absolutely dependent on the presence of normal thymic architecture and cellular composition. These findings are consistent with a checkpoint role for the pre-TCR in regulating the divergence of survival and cell death fates at the CD4-8- to CD4+8+ transition. Further, our data suggest that precursor thymocyte differentiation is induced by other, probably ubiquitous, mechanisms that require the presence of normal thymic cellularity, composition, and architecture.

T cell receptor (TCR)-beta gene recombination: dissociation from cell cycle regulation and developmental progression during T cell ontogeny.

T cell lymphopoiesis involves extensive cell division and differentiation; these must be balanced by export and programmed cell death to maintain thymic homeostasis. Details regarding the nature of these processes, as well as their relationships to each other and to the definitive process of T cell receptor (TCR) gene recombination, are presently emerging. Two widely held concepts are that cell cycle status is inherently and inversely linked to gene recombination and that the outcomes of gene recombination regulate developmental progression. In this study, we analyze TCR-beta recombination and cell cycle status with respect to differentiation during early T cell ontogeny. We find that although differentiation, cell cycle fluctuations, and gene recombination are coincident during normal T cell development, differentiation and cell cycle status are not inherently linked to the recombination process or its products. Rather, recombination appears to occur in parallel with these events as part of a genetically patterned program of development. We propose that the outcome of gene recombination (i.e., TCR expression) may not influence developmental progression per se, but instead serves to perpetuate those developing cells that have been successful in recombination. The potential consequences of this model for the regulation of thymic lymphopoiesis and programmed cell death are discussed.

TCR gene recombination and alpha beta-gamma delta lineage divergence: productive TCR-beta rearrangement is neither exclusive nor preclusive of gamma delta cell development.

Two types of T lymphocytes can be generated intrathymically, distinguishable by either TCR-gamma delta or -alpha beta surface expression. Regulation of the intrathymic divergence of these cells is unresolved, at least in part because thymically derived gamma delta cells have rarely been studied. We used quantitative Southern blotting together with PCR-based cloning/sequencing and restriction fragment length polymorphism to analyze TCR-alpha and -beta gene recombination in thymically derived gamma delta cells. We found that TCR-beta gene recombination is a frequent occurrence in thymic gamma delta cells. Furthermore, not only do complete (V-D-J) TCR-beta gene rearrangements occur in thymic gamma delta cells, but the frequency of in-frame rearrangements is greater than would be predicted based upon random occurrence. In contrast, we show that thymically derived gamma delta cells do not make detectable rearrangements of the TCR-alpha locus. These studies clearly demarcate a point for alpha beta vs gamma delta commitment in the thymus, after TCR-beta but before TCR-alpha gene recombination. Further, while our data support gamma delta lineage commitment as a consequence of successful TCR-gamma and -delta gene rearrangement, we do not find support for a competitive model of lineage commitment, since productive TCR-beta gene rearrangement does not necessarily relegate cells to the alpha beta lineage.