Panel A shows flow cytometry plots and a bar graph comparing the number of total macrophages, including CD169 positive macrophages, CD169 negative MHC 2 positive macrophages, and CD169 negative MHC 2 negative macrophages in control and B16-F10-bearing skin. The bar graph has cell counts on the y-axis and different macrophage types on the x-axis. Panel B presents bar graphs showing the cell counts of CD169 positive macrophages, CD169 negative MHC 2 positive macrophages, and CD169 negative MHC 2 negative macrophages in control and B16-F10-bearing skin. Panel C illustrates the experimental scheme for the depletion of skin resident macrophages before B16-F10 tumor implantation. Panel D includes a line graph showing tumor volumes over time with isotype mAb versus anti-CSF1R antibodies, with tumor volume on the y-axis and days on the x-axis, along with bar graphs for day 7 and 11. Panel E displays bar graphs of the numbers of skin total macrophages, CD169 positive macrophages, CD169 negative MHC 2 positive macrophages, and CD169 negative MHC 2 negative macrophages in tumor-bearing skin under treatment with isotype mAb or anti-CSF1R mAb. Panel F shows a bar graph of the ratio of CD45.2 positive cells in CD169 positive macrophages in normal or B16-F10-bearing skin. Panel G presents flow cytometry plots and bar graphs of the numbers of Tomato positive and negative CD169 positive macrophages, CD169 negative MHC 2 positive macrophages, and CD169 negative MHC 2 negative macrophages in control or B16-F10-bearing skin of Ms4a3cre.tdTomLSL mice.
Skin CD169+ macrophages suppress B16-F10 melanoma growth. (A and B) Representative flow cytometry plots of skin Mϕ (CD45+ CD11b+ CD64+ F4/80+ cells) in control or B16-F10–bearing skin (day 11). The number of total Mϕ, including CD169+ Mϕ, CD169− MHCII+ Mϕ, and CD169− MHCII− Mϕ in control (Ctrl) or B16-F10–bearing skin, were analyzed using flow cytometry (n = 5 for each group, data are shown with mean ± SD, representative of two independent experiments). P value was calculated using Student’s t test. (C) Experimental scheme for the depletion of skin-resident macrophages before B16-F10 tumor implantation. 200 μg isotype mAb or anti-CSF1R mAb was subcutaneously injected on days −3 and −1, and then 1.0 × 105 B16-F10 cells were intradermally/subcutaneously injected into the mouse back skin on day 0. (D) The tumor volumes of B16-F10 melanoma with isotype mAb versus anti-CSF1R antibodies (n = 15 for each group, data are combined from three independent experiments with five mice per group. The results are expressed as the mean with a 95% confidence interval). P value was calculated using two-way ANOVA. (E) The numbers of skin total Mϕ, CD169+ Mϕ, CD169− MHCII+ Mϕ, and CD169– MHCII– Mϕ in the tumor-bearing skin were analyzed using flow cytometry on day 11 under treatment with isotype mAb or anti-CSF1R mAb (n = 4–5 for each group, data are representative of two independent experiments). P value was calculated using Student’s t test. (F) The ratio of CD45.2+ cells in CD169+ Mϕ in normal or B16-F10–bearing skin on day 12 was analyzed by flow cytometry. 1.0 × 105 B16-F10 cells were intradermally/subcutaneously injected into the mouse back skin 10–12 wk after the lethal irradiation, followed by BM cell transfer from CD45.1+ congenic mice (n = 5 for each group, data are representative of two independent experiments). P value was calculated using Student’s t test. (G) Representative flow cytometry plots of CD169+ Mϕ, CD169− MHCII+ Mϕ, CD169– MHCII– Mϕ in control or B16-F10–bearing skin of Ms4a3cre.tdTomLSL mice (day 9). The numbers of Tomato-positive and -negative CD169+ Mϕ, CD169– MHCII+ Mϕ, and CD169– MHCII– Mϕ in control (Ctrl) or B16-F10–bearing skin were analyzed using flow cytometry (n = 4 for each group, data are shown with mean ± SD, representative of two independent experiments). Each circle represents one mouse. P value was calculated using Student’s t test.