Figure 5. LtβR signaling is required for high level PD-L1 expression by medullary LEC, and MAdCAM-1 expression on subcapsular LEC.

a. LNSC were purified by enzymatic digestion of pooled LN and CD45 magnetic bead separation from mice treated for 4 weeks with LtβR-Ig or PBS, and stained with antibodies specific for gp38, CD31, PD-L1, ICAM-1, MAdCAM-1, and LtβR, and analyzed by flow cytometry. Left panel, plots display data gated on CD45^neg gp38⁺ CD31⁺ LEC and subsequently gated on PD-L1 and ICAM-1 co-expressing subpopulations. Numbers indicate percentage of gated population out of total LEC (PD-L1 vs. ICAM-1 2-D plots), or out of the total PD-L1^hi ICAM-1^hi subpopulation (MAdCAM-1 vs. LtβR 2-D plots). Right panel, summary of 3 independent experiments. Data are represented as mean +/− SEM. b. PD-L1 MFI of PD-L1^hi ICAM-1^hi and PD-L1^int ICAM-1^int subpopulations gated on gp38⁺ CD31⁺ LN cells of the indicated mice. Each set of paired samples represents an independent experiment. c. LEC absolute number was calculated from cells that were gated as Dapi^neg, singlets, CD45^neg, gp38⁺, CD31⁺ cells by flow cytometry. Data are represented as mean +/− SEM. d. Analysis of gp38⁺ CD31⁺ LN cells as in (a) in mice treated for 1 week with LtβR-Ig or PBS. Data from 3 independent experiments. e. Left panel, frozen axillary LN sections of indicated mice were stained with antibodies specific for Lyve-1, and PD-L1. Right panel, summary plot of PD-L1 MFI gated on Lyve-1⁺ pixels in the indicated LN location. Data are represented as mean +/− SEM. Significance values represent a comparison of the MFI of PD-L1 staining of Lyve-1⁺ vessels between LtβR-Ig and PBS treated mice of the indicated LN compartment. S = subcapsule C = cortex, M = medulla. Scale bar = 200 µm. f. Left panel, frozen axillary LN sections from indicated mice were stained with antibodies specific for Lyve-1 and MAdCAM-1. Scale bar = 200 µm. Right panel, summary plot of MAdCAM-1 MFI gated on Lyve-1⁺ pixels in the LN subcapsule. MFI calculated as in (c). Staining is representative of multiple fields from 3 independent experiments consisting of 2 separate LN from 3 mice. Data are represented as mean +/− SEM. *p<0.05, **p<0.01, ***p<0.001, ns = not significant. g. Prox1-CreERT2 x LtβR^fl/+ (LtβR-fl/+) and Prox1-CreERT2 x LtβR^fl/fl (LtβR-fl/fl) mice were treated for 2 weeks with a Tamoxifen diet. Upper panel, LNSC were purified as above, stained with antibodies specific for CD45, gp38, CD31, and LtβR and assessed by flow cytometry. Lower panels, summary plots of the representation of PD-L1 and ICAM-1 LN-LEC subpopulations and MAdCAM-1 MFI. Data are represented as mean +/− SEM. ns = not significant.