Fig. 5.

Internalization of stromal LTβR correlates with induction of MAdCAM-1. (A) RelB is necessary for a sustained induction of MAdCAM-1 expression in E14 mLN organ culture treated with an agonistic antibody to LTβR. Flow cytometry analysis of single cell suspensions of wt (left panel) and RelB^−/− (right panel) E14 mLNs in organ cultures for 3 days. Percentages shown in histograms correspond to CD45⁻ stromal cells. Three different stromal cell populations were gated: ICAM-1^neg VCAM-1^neg (I^neg V^neg), I^int V^int, and I^high V^high. Expression levels of MAdCAM-1 for each cell population are shown in histograms. (B) Emergence of the I^high V^high stromal organizer cells in mLNs. Flow cytometry analysis of single cell suspensions of mLNs at E15 and E17 showing the recruitment of CD45⁺ hematopoietic cells and the concomitant phenotypic changes in the CD45⁻ stromal cells. Percentages shown in the histogram correspond to CD45⁻ stromal cells and CD45⁺ hematopoietic cells. At E15, the stromal cell population expressed low levels of ICAM-1 and VCAM-1 (I^int V^int cell population). These cells expressed LTβR but were MAdCAM-1 negative. At E17, the stromal cells have matured and some cells express high levels of ICAM-1 and VCAM-1 (I^high V^high cell population, in red) and MAdCAM-1 but downregulated LTβR cell surface expression. (C) The I^int V^int and I^high V^high cell populations expressed the same levels of LTβR mRNA. I^int V^int and I^high V^high stromal cell populations of mLNs at E18 were sorted and analyzed for LTβR mRNA expression by real-time PCR. Ratios of the gene of interest to the β-actin gene are shown. Results are representative of at least 3 independent experiments.