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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