Figure 2.
Progressively Growing B16-F10 Pulmonary Metastases Cause Local Immunosuppression
(A) Schematic representation of the experimental setup of tumor score and BALF analysis after i.v. injection of syngeneic B16-F10 melanoma cells into C57BL/6 mice. (B) B16-F10 cells (2 × 105 per mouse) were injected intravenously to establish pulmonary metastases, and the amount of tumor tissue in relationship to total lung tissue was investigated via IHC staining of MCR1 at the indicated time points. Three histological lung specimen representing different lung parts were analyzed for each mouse. (C–H) Graphs represent changes in the immune status of the bronchoalveolar space of C57BL/6 mice after i.v. injection of 2 × 105 B16-F10 melanoma cells. (C) Absolute numbers of total leucocytes (CD45.2+) as well as total numbers of macrophages (F4/80+), neutrophils (CD11b+CD11c−Ly-6G+), and T cells (CD3+) as subpopulations of CD45.2+ cells. For schematic representation of the principal gating strategy, see Figure S1. (D) Images represent alveolar macrophages (CD11c+CD11b−Siglec-F+) in the BALF. The left image shows the total number of alveolar macrophages in BALF per mouse as part of CD45.2+ cells. The middle images show the percentage of IL-R4α+ and MHCII+ cells. The right image is a dot plot distribution of these cells on day 19 after tumor implantation. The gating strategy by means of FMO controls is presented in Figure S2. (E) Images represent peripheral macrophages (CD11b+CD11c−F4/80+). (F–H) Images represent (F) NK cells (CD3−CD49b+), (G) T helper cells (CD3+CD4+), and (H) T killer cells (CD3+CD8+) among CD45.2+ cells in BALFs. As described for (D), the left images show the total number of appropriate cells per mouse, while the middle images show percentages of cells expressing indicated activation markers and the right images are dot plot distributions of cells with specific markers on day 19 after B16-F10 implantation. Note that the number of peripheral macrophages and NK cells in BALFs at early stages of tumor growth was very low, and no reliable data for cells expressing specific markers could be obtained. Data are expressed as means ± SEM, with n = 4-6 animals per group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (Kruskal-Wallis test with a Dunn’s multiple comparison test).