Fig. 2.
Increased immune checkpoint expression in BCC tumors. (A) qPCR analysis of mouse skin (n for Pd‐1 and Cd226: Ptchf/f = 5 and PtchΔep = 4, for Pd‐l1 and Tim3: Ptchf/f = 6 and PtchΔep = 6, for Pd‐l2 and CD96: Ptchf/f = 5 and PtchΔep = 5, and for Tigit: Ptchf/f = 5 and PtchΔep = 6). (B) Representative flow cytometry plot showing PD‐1 expression on CD3+ T cells in mouse skin (upper panel) and flow cytometry analysis of PD‐1 expression on CD3+ T cells (lower panel; Ptchf/f n = 4 and PtchΔep n = 6). Cells were pregated for CD3. (C) Immunofluorescence analysis of murine ear skin stained for PD‐1 (green) and DAPI (blue). The arrows highlight PD‐1+ cells (scale bar 20 µm). (D) Flow cytometry analysis of PD‐L1 expression on CD45− Sca‐1+ (Ptchf/f n = 5 and PtchΔep n = 7) and CD49f+ (Ptchf/f n = 6 and PtchΔep n = 7) keratinocytes in mouse skin. (E) Treatment schedule of PD‐1 blocking in previously TAM‐treated K14CreERT;Ptchf/f BCC mice. (F) Representative images of H&E staining of ear skin of PtchΔep mice treated with anti‐PD‐1 blocking antibody or untreated (n = 4 per group). The dashed lines mark the tumor area (scale bar 100 µm). (G) Representative immunohistochemical stainings of human BCC skin sections for PD‐L1 (brown) alone and together with CD8 (red) nuclei are stained in blue. The samples were divided by PD‐L1 low (n = 5) and high (n = 3) expression (scale bar 20 µm). (H) Quantification and statistical analysis of the CD8 and CD8 plus PD‐L1 and PD‐1 expression from PD‐L1 low (n = 5) and high (n = 3) BCC. D, dermis; E, epidermis; T, tumor mass. Statistical analysis was performed with Student's t test except for (H), which was analyzed with the Levene test to prior t test. For (H), the whiskers show the minimum and maximum of data points. *P < 0.05, **P < 0.01 and ***P < 0.001.