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Mentions: T lymphocyte development, can be studied in vitro by coculturing haematopoietic progenitors with OP9 stromal cells expressing Notch ligand Delta-like-1 (OP9-DL1). We applied this system together with Eva1 lentiviral RNA interference (RNAi) to evaluate the role of EVA1 in T cell differentiation . Haematopoietic precursors isolated from E14,5 fetal liver (FL) by cell sorting (Lin−; Sca1+; c-Kit+)(LSK) were used. Interference with Eva1 was performed by infection with lentiviral vectors (see material and methods for details). The efficiency of RNA interference was assessed by qRT-PCR and immunofluorescence analysis (IF). As shown in Figure 1C (upper panel), LSK cells interfered for Eva1 (EVAi) showed a strong reduction of Eva1 expression, while LSK infected with control lentiviral vector (CT) showed an expression level of Eva1 comparable to uninfected LSK-WT cells. Interference was confirmed by IF (Fig. 1C, lower panel) using a rabbit polyclonal serum anti-EVA1. We tested three different interfering sequences obtaining comparable results (data not shown). To address whether EVA1 depletion had an effect on T cell development, progressive phenotype acquisition based on CD44/CD25 and CD4/CD8 expression, was evaluated by flow cytometry in cocultures experiments. As described , LSK-CT cocultured with OP9-GFP cells did not show the CD44/CD25 progression and did not give rise to T cells (Fig. 2A, left and right panels). Otherwise, after 4 days of coculture, LSK-CT cells cultured on OP9-DL1 cells showed a differential surface expression of CD44/CD25 molecules (Fig. 2A, left panel). The progression in CD44/CD25 maturation pathway was observed until 18 days of coculture (Fig. 2A, left panel). This progression correspond to CD4 and CD8 expression (Fig. 2A, right panel). LSK-CT cells gave rise to CD4+CD8+ immature double positive (DP) T cells after 8 days of coculture (Fig. 2A, right panel), with an increase of DP cells after 18 days of coculturing (Fig. 2A, right panel). The temporal kinetics of LSK-CT differentiation cocultured with OP9-DL1 cells was similar to that observed with LSK-WT cells (data not shown). A delayed progression into the CD44/CD25 maturation pathway was observed when LSK-EVAi cells were cultured on OP9-DL1 cells and compared to LSK-CT progression (Fig. 2A, left panel). After 18 days, LSK-EVAi cells were arrested at DN2-DN3 stages and failed to generate DP cells (Fig. 2A, right panel). These data confirmed that Eva1 upregulation occurs at DN1-DN3 transition during physiological thymocyte development and maturation in vitro.
Lymphoid EVA1 Expression Is Required for DN1-DN3 Thymocytes Transition
Bottom Line: Gene expression occurring during T lymphocyte differentiation must be coordinated in a spatio-temporal fashion; one way in which this is achieved is through the regulation by cell-cell adhesion and interactions.Fetal liver derived haematopoietic progenitors depleted of Eva1, displayed a delayed DN1-DN3 transition and failed to generate CD4CD8 double positive T cells in OP9-DL1 coculture system.Similarly, Rag2-gamma c double knock out mice, transplanted with Eva1 depleted haematopoietic progenitors displayed a 10-fold reduction in thymus reconstitution and a time delayed thymocytes maturation compared to controls.
Affiliation: San Raffaele Biomedical Science Park Foundation, Rome, Italy.
Background: Thymus organogenesis and T lymphocyte development are accomplished together during fetal life. Proper development and maintenance of thymus architecture depend on signals generated by a sustained crosstalk between developing thymocytes and stromal elements. Any maturation impairment occurring in either cellular component leads to an aberrant thymic development. Gene expression occurring during T lymphocyte differentiation must be coordinated in a spatio-temporal fashion; one way in which this is achieved is through the regulation by cell-cell adhesion and interactions.
Principal findings: We examined the role played by Epithelial V-like Antigen 1 (EVA1), an Ig adhesion molecule expressed on thymus epithelial cells (TEC) and immature thymocytes, in T cell development by employing RNA interference in vitro and in vivo models. Fetal liver derived haematopoietic progenitors depleted of Eva1, displayed a delayed DN1-DN3 transition and failed to generate CD4CD8 double positive T cells in OP9-DL1 coculture system. In addition, we could observe a coordinated Eva1 up-regulation in stromal and haematopoietic cells in coculture control experiments, suggesting a possible EVA1 involvement in TEC-haematopoietic cells crosstalk mechanisms. Similarly, Rag2-gamma c double knock out mice, transplanted with Eva1 depleted haematopoietic progenitors displayed a 10-fold reduction in thymus reconstitution and a time delayed thymocytes maturation compared to controls.
Conclusions: Our findings show that modulation of Eva1 expression in thymocytes is crucial for lymphocyte physiological developmental progression and stromal differentiation.
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