Abstract

IL-7 is a non-redundant cytokine in T cell development. We studied the role of IL-7 in early T-cell development using a model of transgenic (Tg) mice with the murine IL-7 gene under control of the lck proximal promoter. At high IL-7 over-expression (x39 fold increase at day 1 in total thymic tissue), we observed a disruption of TCRαβ development along with increased B cell development in the thymus (7- to 13-fold increase) (El Kassar, Blood, 2004). In order to further explore abnormal T and B cell thymic development in these mice, we first confirmed that they both arise in parallel and were non-cell autonomous, by in vivo injection of neutralizing anti-IL-7 MAb and mixed bone marrow chimera experiments. Using a six color flow cytometry analysis, we found a dramatic decrease of the early thymocyte progenitors (ETPs, linCD44+CD25c-kithiIL-7R−/lo) in the adult Tg mice (x4.7 fold decrease). LinCD44+CD25c-kit+ thymocytes were sorted and cultured on OP9 and OP9 delta-like1 (OP9-DL1) stromal cells (kindly provided by Pr Zuniga Pflucker). At day 14, we observed an important decrease of T cell development (54% vs. 1% of DP cells) and an increase of NK cells (x5 fold increase) in the Tg-derived DN1 cell culture. DN2 (LinCD44+CD25c-kit+) Tg thymocytes showed the same, but less dramatic abnormalities. While DN1 progenitors developed effectively into B220+CD19+ cells on OP9 stromal cells, no B cell development was observed on OP-DL stromal cells from DN1-Tg derived progenitors or by addition of increasingly high doses of IL-7 (x10, x40, x160) to normal B6-derived DN1 progenitors. Instead, a block of T-cell development was observed with increased IL-7. We hypothesized a down regulation of Notch signaling by IL-7 over-expression and analyzed by FACS Notch expression in the DN thymocytes. By staining the intra-cellular part of Notch cleaved after Notch 1/Notch ligand activation, Tg-derived DN2 cells showed decreased Notch signaling. More importantly, HES expression was decreased in the DN2, DN3 and DN4 fractions by semi-quantitative PCR. Sorted Pro/Pre B cells from Tg thymi showed TCR Dβ1-Jβ1 rearrangement indicating their T specific origin, in opposition to Pro/Pre B cells sorted from the bone marrow of the same mice. We suggest that more than one immature progenitor seeds the thymus from the bone marrow. While ETPs had T and NK proliferative capacity, another thymic progenitor with B potential may be responsible for thymic B cell development in normal and IL-7 Tg mice. Finally, IL-7 over-expression may induce a decreased Notch signaling in thymic progenitors, inducing a switch of T vs. B lineage development.

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