Silva Santos, Bruno Miguel Carvalho; (2002) T cell development and lineage commitment: Studies based on differential gene expression in thymocyte subsets. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

T lymphocytes develop primarily in the thymus, where lymphoid progenitors imported from foetal liver or adult bone marrow go through a series of differentiation events that produce mature T cells bearing antigen-specific T cell receptors (TCR). Early thymocyte development (before TCR expression) is marked by two crucial events: [alpha][beta] versus [gamma][delta] T cell lineage commitment; and "[beta]-selection". The two T cell lineages derive from a common thymic progenitor and are defined by the TCR isotype expressed on the cell surface, [alpha][beta] or [gamma][delta]. "[beta]-selection" consists of a checkpoint in the differentiation of the [alpha][beta] lineage, in which only precursors that receive signals from the pre-TCR - a complex made of a newly synthesised TCR[beta] chain and the invariant protein pT - are selected for further maturation. Since very little was known about the genetic programme that accompanies these two processes, we used cDNA-RDA (Representation Difference Analysis) to identify genes differentially expressed in thymocyte subsets representative of distinct lineages or developmental stages. The ICER isoform of the CREM gene was identified as differentially expressed between lineage-committed thymic [alpha][beta] and [gamma][delta] T cell populations. Although thymic development was unperturbed in CREM/ICER-/- mice, we demonstrate that subsequent to the DN4 stage of thymocyte differentiation, ICER is a robust marker of the [gamma][delta] T cell lineage. ICER expression is not observed in [alpha][beta] -committed DP or SP thymocytes, or in [alpha][beta] T cells from the lymph node and spleen. Furthermore, we show that ICER expression is a characteristic of developmental lineage rather than the type of TCR that is expressed, which supports a non-instructive mechanism for the lineage divergence. In addition, the analysis of ICER expression in subsets of less well characterised intestinal intraepithelial lymphocytes (IELs) allowed us to propose a refinement to the conventional [alpha][beta]/[gamma][delta] classification of T cells that incorporates TCR[alpha][beta](+)CD8[alpha][alpha](+) IELs as having a "[gamma][delta]-like" profile. Surprisingly (for a [gamma[delta] lineage marker), ICER expression is severely impaired in the thymus of pT[alpha]-/- and TCR[beta]-/- mice, suggesting a genetic link between pre-TCR and ICER expression. Moreover, ICER expression can be induced in pre-TCR- deficient pre-T cells by CD3 signalling, and this induction is dependent on an intact MAPK pathway. These data suggest that ICER is a downstream target of pre-TCR signalling in pre-T cells. Consistent with this, ICER(+) pre-T cells have the phenotype and developmental behaviour of [beta]-selected thymocytes. However, in the [gamma][delta] lineage, ICER expression is not a direct consequence of pre- TCR signalling, since this complex is absent from the vast majority of [gamma][delta] cells. We provide evidence for the existence, in a normal thymus, of a trans-induction mechanism by which [beta]-selected thymocytes (DP cells in particular) influence gene expression and the physiology of [gamma][delta] cells. This novel mechanism constitutes the first cross-talk reported for the [alpha][beta] and [gamma][delta] lineages during their thymic differentiation.

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