Assessment of IL-7 expression in human LN LECs. (A) Fetal LN sections were stained with fluorescently labeled antibodies against LyveI, MadCAM-1, and CD3. (B) Adult LN sections were stained with antibodies against LyveI, VCAM-1, MadCAM-1, and Pdpn. LyveI⁺Pdpn⁺ LECs (blue in left panel and green in right panel) are indicated in the subcapsular sinus (arrows) and LN medulla (*). (C) CD31⁺Pdpn⁺ LECs, CD31⁻Pdpn⁺ FRCs, and CD31⁺Pdpn⁻ BECs from human fetal mesenteric LNs were sorted by flow cytometry. IL-7 expression levels were determined by quantitative RT-PCR. Values indicate mean ± SEM from triplicates, representative results from 1 of 2 independent sorting experiments. (D) Human LN LECs were cultivated and analyzed for IL-7 mRNA expression by quantitative RT-PCR. Cell culture supernatants were collected after 48 hours and analyzed for IL-7 protein by ELISA (right graph). Measurements were carried out in triplicates (mean ± SEM). (E) MACS-isolated human naive CD4⁺ T cells (2 × 10⁵) were cocultured with human LECs, HUVECs, or supernatant from human LEC cultures in the presence and absence of neutralizing anti–IL-7 antibody. After 48 hours, T-cell survival was analyzed by flow cytometry and is displayed as difference (Δ) to medium control. Values indicate mean ± SEM from triplicates, representative results from 1 of 2 independent experiments; *P < .05.