Figure 5.

Lymphatic podoplanin regulates abundance of medullary sinus macrophages (MSMs) in tumor-draining lymph LNs. (a) Schematic representation of the experimental set-up. Pdpn^fl/fl x Prox1-CreER^T2 mice were treated for 5 consecutive days with tamoxifen. Cre− littermates served as controls. Subsequently, B16F10 cells were implanted intradermally, tumor growth was monitored, and tumor-draining LNs (inguinal and axillary) were analyzed by flow cytometry on day 14. (b) Representative LEC gating in Cre− and Cre+ mice. Stromal cells (pregated as CD45− living singlets) were stained for podoplanin and CD31 to detect FRCs and endothelial cells, respectively. Subsequently, LECs were distinguished from BECs with a combination of CD41 (Itga2b) and ESAM. (c,d) Representative histogram (c) and quantification (d) of podoplanin expression in LN stromal cells (N = 9 Cre−/7 Cre+). (e) Number of LN stromal cells in tumor-draining LNs determined by flow cytometry (N = 9 Cre−/7 Cre+). (f,g) Representative images (f) and quantification (g) of CD169+ macrophages in subcapsular and medullary sinuses of tumor-draining LNs in Cre− and Cre+ mice (N = 7/group for subcapsular and 5/group for medullary sinus). (h–j) Representative flow cytometry gating ((h), pregated for CD11b+ living singlets), absolute quantification (i), and relative quantification (j) of LN macrophage subsets medullary cord macrophages (MCMs), MSMs and subcapsular sinus macrophages (SSMs) (N = 9 Cre−/6 Cre+). * p < 0.05, **** p < 0.0001, unpaired t-test.