Abstract
Normal T-cell differentiation requires a complex regulatory network which supports a series of maturation steps, including lineage commitment, T-cell receptor (TCR) gene rearrangement, and thymic positive and negative selection. However, the underlying molecular mechanisms are difficult to assess due to limited T-cell models. Here we explore the use of the pro-T-cell line P5424 to study early T-cell differentiation. Stimulation of P5424 cells by the calcium ionophore ionomycin together with PMA resulted in gene regulation of T-cell differentiation and activation markers, partially mimicking the CD4-CD8- double negative (DN) to double positive (DP) transition and some aspects of subsequent T-cell maturation and activation. Global analysis of gene expression, along with kinetic experiments, revealed a significant association between the dynamic expression of coding genes and neighbor lncRNAs including many newly-discovered transcripts, thus suggesting potential co-regulation. CRISPR/Cas9-mediated genetic deletion of Robnr, an inducible lncRNA located downstream of the anti-apoptotic gene Bcl2, demonstrated a critical role of the Robnr locus in the induction of Bcl2. Thus, the pro-T-cell line P5424 is a powerful model system to characterize regulatory networks involved in early T-cell differentiation and maturation.
Highlights
T lymphocytes are one of the main players of the adaptive immunity
Based on the expression level of the Tcra gene, we determined that treatment with 10 ng/ ml of phorbol 12-myristate 13-acetate (PMA) and 0.5 μg/ml of ionomycin for 4 h resulted in the highest gene induction (Supplementary Fig. 1A)
To further validate these findings, we analyzed the expression of the human (h)CD25 in a stable transfected P5424 cell line, where hCD25 is under the control of the mouse Ptcra promoter[42] (Supplementary Fig. 1C)
Summary
T lymphocytes are one of the main players of the adaptive immunity. T-cell development in the thymus requires temporally regulated rearrangements of the T-cell receptor (Tcr) genes and a series of selection events, whereby newly assembled TCR complexes signal for cell survival, proliferation and differentiation processes[1,2]. The expression of a functionally rearranged Tcra gene leads to the formation of a variable TCRαβ heterodimer and, to the selection of TCRαβ expressing cells which will terminally differentiate into CD4+ or CD8+ single positive (SP) T cells Disruptions of these genetic and epigenetic processes might result in oncogenic transformation of T-cell precursors (i.e. leukemia and lymphoma7,8) or immune-related pathologies[9]. Genetic inactivation of the PMA/ionomycin inducible lncRNA XLOC_000895 (Robnr), located downstream of the Bcl[2] gene, resulted in impaired Bcl[2] activation, revealing a critical regulator of the Bcl[2] locus and highlighting the usefulness of the P5424 pro-T-cell line to dissect the molecular basis of T-cell regulatory networks
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