Lymphocytes sequestration in LN limits blood lymphocytes access to the LN parenchyma. RAG-2°^/°ubiquitin-GFP chimera were irradiated and reconstituted with either WT bone marrow cells or a mixture of bone marrow cells isolated from RAG-2°^/°WT mice (20%) and ubiquitin-Cre Esr1 × S1P1^flox/− mice (80%). Reconstituted chimeras were treated or not with tamoxifen and injected with 10 millions of CMTMR labeled lymphocytes (green). One hour later, LNs were harvested, sectioned, stained for PNAd (red) and analyzed by confocal microscopy (A). Arrowheads indicate HEVs in which many transferred cells accumulate. (B) Densities of transferred cells inside and outside PNAd⁺ HEVs were calculated for each analyzed section and presented as a ratio. (C) A typical HEV imaged in control and tamoxifen-treated ubiquitin-Cre Esr1 × S1P1^flox/− is shown. Data are representative of 3 different experiments (2 or 3 mice per experiment, minimum of 3 LNs per mouse).