Fig 2: Peritumoral LECs upregulate MHC-II, PD-L1, and various co-inhibitory molecules compared to naïve dermal LECs. (A) Schematic of experimental design analyzing naïve dermal LECs in the ear and peritumoral LECs of the same mice. (B) Quantification of MHC-II and PD-L1 positivity in naïve and peritumoral LECs (CD45- podoplanin+ CD31+) at day 7, 14, and 21 post B16F10 tumor inoculation in WT (C57/BL6) mice. (C) Quantification of MHC-II and PD-L1 positivity in naïve and peritumoral LECs at day 14 post E0771 tumor inoculation in WT mice. (D) Quantification of MHC-II and PD-L1 positivity in naïve and peritumoral LECs in MMTV-PyMT mice with spontaneous mammary tumors. (E) Quantification of PD-L2, HVEM, and CD48 positivity in naïve and peritumoral LECs at day 21 post B16F10 tumor inoculation in WT mice. (F) Representative immunofluorescence image stained for LYVE-1 (white) and MHC-II (green) in peritumoral and skin margin lymphatics in WT mice at day 14 post B16F10 inoculation. DAPI nuclear staining in blue. Scale bars, 100 µm (left), 20µm (top right and bottom right). Statistical significance assessed using paired student’s t test on data from at least 3 biological replicates; error represented as SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig 3: IFN? induces upregulation of MHC-II in LECs in vitro and in vivo in the tumor periphery. (A–D) Relative mRNA expression of HLA-DRA, HLA-DM, PD-L1, LYVE-1 in cultured human dermal LECs after 24-h treatment with (A) IFN? (100 ng/mL), (B) VEGF-C (100 ng/mL), (C) IL-2 (10 ng/mL) and (D) TNF-a (10 ng/mL). (E) Representative FACS histogram plot and (F) quantification of MHC-II and PD-L1 positive human dermal LECs in culture after 24-hour treatment with IFN? (100 ng/mL) as measured by flow cytometry. (G) Representative FACS histogram plots and (H) quantification of MHC-II and PD-L1 positive peritumoral LECs in WT mice treated with IFN? neutralizing antibody or IgG isotype control at day 14 post tumor inoculation. Statistical significance assessed using unpaired student’s t test on data from at least 3 biological replicates for cell culture studies and at least 4 biological replicates for mouse experiments; statistical analysis not performed on (B–D) for which at least 2 biological replicates were used; error represented as SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001. n.d, not detected mRNA in treated group.