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Although the mechanisms of cross-talk that regulate the hematopoietic and epithelial compartments of the thymus are well established, the interactions of these compartments with the thymic endothelium have been largely ignored. Current understanding of the thymic vasculature is based on studies of adult thymus. We show that the neonatal period represents a unique phase of thymic growth and differentiation, marked by endothelium that is organized as primitive, dense networks of capillaries dependent on vascular endothelial growth factor (VEGF). VEGF dependence in neonates is mediated by significantly higher levels of both VEGF production and endothelial VEGF receptor 2 (VEGF-R2) expression than in the adult thymus. VEGF is expressed locally in the neonatal thymus by immature, CD4−CD8− “double negative” (DN) thymocytes and thymic epithelium. Relative to adult thymus, the neonatal thymus has greater thymocyte proliferation, and a predominance of immature thymocytes and cortical thymic epithelial cells (cTECs). Inhibition of VEGF signaling during the neonatal period results in rapid loss of the dense capillaries in the thymus and a marked reduction in the number of thymocytes. These data demonstrate that, during the early postnatal period, VEGF mediates cross-talk between the thymocyte and endothelial compartments of the thymus.

The intrathymic differentiation events leading to the development and export of mature T cells tolerant to self yet capable of responding to foreign peptide antigen in the context of self-MHC are clearly both dynamic and complex. The changing phenotype of the developing thymocyte as it migrates through and interacts with the heterogeneous thymic microenvironment and the intracellular signalling events associated with such interactions are being extensively studied, yet many aspects remain poorly defined, such as the precise relationship between stromal cells and thymic selection. Positive and negative selection are crucial events in the development of T cells, leading to a diverse yet non-autoreactive immune system. A breakdown in either of these processes could lead to either a reduced T cell repertoire or the escape into the periphery of autoreactive T cells - both clearly having deleterious consequences for the health of the individual. This review aims to summarise the current status of research in thymic positive selection with emphasis on the role of different cell types and peptides.

The thymus organ is composed of a three-dimensional (3D) network of adjoining epithelium and stromal cells. Bone marrow-derived T cell precursors, upon entering the thymus, interact with and migrate through this cellular network as they differentiate and mature. An essential component of the stroma is the thymic epithelial cells (TEC), which play a vital role in T cell development and induction of self-tolerance for adaptive immunity. TEC can be isolated from the embryonic and adult thymus by a series of gentle enzymatic digestions and characterized into discrete subpopulations based on their expression of surface markers by flow cytometry. Enrichment of adult TEC can be achieved by depletion of hematopoietic cells, allowing sufficient numbers to be purified for subsequent functional and molecular analysis. Although monolayer cultures have been used to study TEC phenotype and T cell interaction, methods that mimic the 3D thymic microenvironment, such as fetal and reaggregate thymic organ cultures, are more accurate for the analysis of TEC function and support more complete T cell development. Herein, we describe methods for the efficient isolation and enrichment of TEC for downstream analyses as well as the reaggregation of embryonic progenitor epithelium to form a functional thymus graft under the kidney capsule.

The thymus is colonized by circulating progenitor cells that differentiate into mature T cells under the influence of the thymic microenvironment. We report here the cloning and function of the avian thymocyte Ag ChT1, a member of the Ig superfamily with one V-like and one C2-like domain. ChT1-positive embryonic bone marrow cells coexpressing c-kit give rise to mature T cells upon intrathymic cell transfer. ChT1-specific Ab inhibits T cell differentiation in embryonic thymic organ cultures and in thymocyte precursor cocultures on stromal cells. Thus, we provide clear evidence that ChT1 is a novel Ag on early T cell progenitors that plays an important role in the early stages of T cell development.

Expression of a single Ag receptor on lymphocytes is maintained via allelic exclusion that generates cells with a clonal receptor repertoire. We show in normal mice and mice expressing functionally rearranged TCR alphabeta transgenes that allelic exclusion at the TCR alpha locus is not operational in immature thymocytes, whereas most mature T cells express a single TCRV alpha-chain. TCRV alpha allelic exclusion in mature thymocytes is regulated through a CD45 tyrosine phosphatase-mediated signal during positive selection. Using functional and genetic systems for selection of immature double TCRV alpha+ thymocytes, we show that peptide-specific ligand recognition provides the signal for allelic exclusion, i.e., mature T cells maintain expression of the ligand-specific TCRV alpha-chain, but lose the nonfunctional receptor. Whereas activation of TCRV beta-chains or CD3epsilon leads to receptor internalization, TCRV alpha ligation promotes retention of the TCR on the cell surface. Although both TCRV alpha- and TCRV beta-chains trigger phosphotyrosine signaling, only the TCRV beta-chain mediates membrane recruitment of the GTPase dynamin. These data indicate that TCRV alpha-directed signals for positive selection control allelic exclusion in T cells, and that developmental signals can select for single receptor usage.

Expression of a single Ag receptor on lymphocytes is maintained via allelic exclusion that generates cells with a clonal receptor repertoire. We show in normal mice and mice expressing functionally rearranged TCRαβ transgenes that allelic exclusion at the TCRα locus is not operational in immature thymocytes, whereas most mature T cells express a single TCRVα-chain. TCRVα allelic exclusion in mature thymocytes is regulated through a CD45 tyrosine phosphatase-mediated signal during positive selection. Using functional and genetic systems for selection of immature double TCRVα+ thymocytes, we show that peptide-specific ligand recognition provides the signal for allelic exclusion, i.e., mature T cells maintain expression of the ligand-specific TCRVα-chain, but lose the nonfunctional receptor. Whereas activation of TCRVβ-chains or CD3ε leads to receptor internalization, TCRVα ligation promotes retention of the TCR on the cell surface. Although both TCRVα- and TCRVβ-chains trigger phosphotyrosine signaling, only the TCRVβ-chain mediates membrane recruitment of the GTPase dynamin. These data indicate that TCRVα-directed signals for positive selection control allelic exclusion in T cells, and that developmental signals can select for single receptor usage.