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Lymphoid EVA1 Expression Is Required for DN1-DN3 Thymocytes Transition

Iacovelli S, Iosue I, Di Cesare S, Guttinger M - PLoS ONE (2009)

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.

ABSTRACT

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.

In vitro T cell development with LSK-EVAi cells.(A) Time course of T cell differentiation of LSK cells infected with a non-interfering (LSK-CT) or with an Eva1 interfering lentivirus (LSK-EVAi) respectively, cocultured with either OP9-GFP control cells or OP9-DL1. Flow cytometric analysis of CD25/CD44 (left panel) and CD4/CD8 (right panel) stainings revealed that LSK-EVAi cells had a delayed DN1-DN3 transition and failed to generate CD4 and CD8 cells in OP9-DL1 cocultures. (B) Time course of Eva1 expression in both haematopoietic (left panels) and stromal (right panels) cells, respectively. Cocolture combinations are indicated. No variations in EVA1 expression are observed in either cell type, from cocoltures that do not sustain T cell development (LSK-CT/OP9 and LSK-EVAi/DL1). Eva1 is strongly up-regulated at 4 and 8 days of LSK-CT/DL1 coculture, during DN1-DN3 transition. Note: scale amplitude in Eva1 real-time RT-PCR in haematopoietic cells. Eva1 is concomitantly up-regulated in stromal OP9-DL1 counterpart too. (C) Right panel: time course of Eva1 expression in haematopoietic cells coltured on OP9 cells overexpressing EVA1. Left panel: EVA1 expression in OP9 and OP9-EVA cells, respectively.
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pone-0007586-g002: In vitro T cell development with LSK-EVAi cells.(A) Time course of T cell differentiation of LSK cells infected with a non-interfering (LSK-CT) or with an Eva1 interfering lentivirus (LSK-EVAi) respectively, cocultured with either OP9-GFP control cells or OP9-DL1. Flow cytometric analysis of CD25/CD44 (left panel) and CD4/CD8 (right panel) stainings revealed that LSK-EVAi cells had a delayed DN1-DN3 transition and failed to generate CD4 and CD8 cells in OP9-DL1 cocultures. (B) Time course of Eva1 expression in both haematopoietic (left panels) and stromal (right panels) cells, respectively. Cocolture combinations are indicated. No variations in EVA1 expression are observed in either cell type, from cocoltures that do not sustain T cell development (LSK-CT/OP9 and LSK-EVAi/DL1). Eva1 is strongly up-regulated at 4 and 8 days of LSK-CT/DL1 coculture, during DN1-DN3 transition. Note: scale amplitude in Eva1 real-time RT-PCR in haematopoietic cells. Eva1 is concomitantly up-regulated in stromal OP9-DL1 counterpart too. (C) Right panel: time course of Eva1 expression in haematopoietic cells coltured on OP9 cells overexpressing EVA1. Left panel: EVA1 expression in OP9 and OP9-EVA cells, respectively.

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 [14]. 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 [14], 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.

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Lymphoid EVA1 Expression Is Required for DN1-DN3 Thymocytes Transition

Iacovelli S, Iosue I, Di Cesare S, Guttinger M - PLoS ONE (2009)

In vitro T cell development with LSK-EVAi cells.(A) Time course of T cell differentiation of LSK cells infected with a non-interfering (LSK-CT) or with an Eva1 interfering lentivirus (LSK-EVAi) respectively, cocultured with either OP9-GFP control cells or OP9-DL1. Flow cytometric analysis of CD25/CD44 (left panel) and CD4/CD8 (right panel) stainings revealed that LSK-EVAi cells had a delayed DN1-DN3 transition and failed to generate CD4 and CD8 cells in OP9-DL1 cocultures. (B) Time course of Eva1 expression in both haematopoietic (left panels) and stromal (right panels) cells, respectively. Cocolture combinations are indicated. No variations in EVA1 expression are observed in either cell type, from cocoltures that do not sustain T cell development (LSK-CT/OP9 and LSK-EVAi/DL1). Eva1 is strongly up-regulated at 4 and 8 days of LSK-CT/DL1 coculture, during DN1-DN3 transition. Note: scale amplitude in Eva1 real-time RT-PCR in haematopoietic cells. Eva1 is concomitantly up-regulated in stromal OP9-DL1 counterpart too. (C) Right panel: time course of Eva1 expression in haematopoietic cells coltured on OP9 cells overexpressing EVA1. Left panel: EVA1 expression in OP9 and OP9-EVA cells, respectively.
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pone-0007586-g002: In vitro T cell development with LSK-EVAi cells.(A) Time course of T cell differentiation of LSK cells infected with a non-interfering (LSK-CT) or with an Eva1 interfering lentivirus (LSK-EVAi) respectively, cocultured with either OP9-GFP control cells or OP9-DL1. Flow cytometric analysis of CD25/CD44 (left panel) and CD4/CD8 (right panel) stainings revealed that LSK-EVAi cells had a delayed DN1-DN3 transition and failed to generate CD4 and CD8 cells in OP9-DL1 cocultures. (B) Time course of Eva1 expression in both haematopoietic (left panels) and stromal (right panels) cells, respectively. Cocolture combinations are indicated. No variations in EVA1 expression are observed in either cell type, from cocoltures that do not sustain T cell development (LSK-CT/OP9 and LSK-EVAi/DL1). Eva1 is strongly up-regulated at 4 and 8 days of LSK-CT/DL1 coculture, during DN1-DN3 transition. Note: scale amplitude in Eva1 real-time RT-PCR in haematopoietic cells. Eva1 is concomitantly up-regulated in stromal OP9-DL1 counterpart too. (C) Right panel: time course of Eva1 expression in haematopoietic cells coltured on OP9 cells overexpressing EVA1. Left panel: EVA1 expression in OP9 and OP9-EVA cells, respectively.
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 [14]. 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 [14], 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.

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.

ABSTRACT

Background:

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.

View Similar Images In: Results  - Collection
View Article: Pubmed Central -  PubMed
Show All Figures - Show MeSH
getmorefigures.php?pmc=2761490&rFormat=json&query=null&req=5