Figure 3.
Figure 3. Overexpression of chemerin increases the number and alters the make up of TILs. (A) CD45+ leukocyte infiltration (percentage of viable cells) from chemerin-expressing versus control tumors excised on day 17 and analyzed by flow cytometry. *, P < 0.05 by two-tailed Student’s t test. (B) Immunofluorescence images illustrate CD45+ cell infiltrates (arrows) in chemerin-expressing melanomas excised at day 9. Bars, 25 µm. (C) Log2 ratio of TIL subset frequency in chemerin-expressing versus control tumors as calculated from FACS analyses of pDCs (Lin−CD11cintB220+PDCA1+), conventional DCs (cDCs; Lin−CD11chiB220−), CD4 (CD3+CD4+) T cells, CD8 (CD3+CD8+) T cells, total T cells (CD3+CD4+CD8+), NK cells (CD3−NK1.1+), CD11b (Lin−CD11b+) monocyte/macrophages, MDSCs (Lin−CD11b+GR1+), and CD19+ B cells (CD3−CD19+). (D) Ratio of NK or total T cells to MDSCs or pDCs in tumors. (E) Absolute numbers of NK and T cells per 10,000 total tumor cells analyzed by FACS from control or chemerin-expressing tumors. Results are from a representative experiment. (A, D, and E) Graphs show mean ± SEM with four or more mice per group. (F) Control (n = 5) or chemerin-expressing B16 (n = 5) was orthotopically implanted into C57BL/6 wild-type mice. Anti–Asialo GM1 depleting antibody was used to deplete NK cells in mice bearing chemerin-expressing tumors (NK depletion; n = 8). *, P < 0.05 comparing tumor size (mean ± SEM) in control or chemerin-expressing versus NK depletion by two-tailed Student’s t test. Data are representative of three experiments. (G) Chemerin-expressing B16 cells were orthotopically implanted in C57BL/6 wild-type and RAG1 KO mice (RAG KO; n = 9) and compared with control B16 cells implanted in WT mice. *, P < 0.05 comparing tumor size (mean ± SEM) in chemerin-expressing or RAG KO versus control by two-tailed Student’s t test. Data are representative of two experiments. (H and I) Control (H) or chemerin-expressing (I) B16 cells were implanted into WT or CMKLR1 KO mice (n = 5/group), and tumor growth was assessed. *, P < 0.05 comparing tumor size (mean ± SEM) in WT versus CMKLR1 KO with implanted chemerin-expressing tumors by two-tailed Student’s t test.

Overexpression of chemerin increases the number and alters the make up of TILs. (A) CD45+ leukocyte infiltration (percentage of viable cells) from chemerin-expressing versus control tumors excised on day 17 and analyzed by flow cytometry. *, P < 0.05 by two-tailed Student’s t test. (B) Immunofluorescence images illustrate CD45+ cell infiltrates (arrows) in chemerin-expressing melanomas excised at day 9. Bars, 25 µm. (C) Log2 ratio of TIL subset frequency in chemerin-expressing versus control tumors as calculated from FACS analyses of pDCs (Lin−CD11cintB220+PDCA1+), conventional DCs (cDCs; Lin−CD11chiB220−), CD4 (CD3+CD4+) T cells, CD8 (CD3+CD8+) T cells, total T cells (CD3+CD4+CD8+), NK cells (CD3−NK1.1+), CD11b (Lin−CD11b+) monocyte/macrophages, MDSCs (Lin−CD11b+GR1+), and CD19+ B cells (CD3−CD19+). (D) Ratio of NK or total T cells to MDSCs or pDCs in tumors. (E) Absolute numbers of NK and T cells per 10,000 total tumor cells analyzed by FACS from control or chemerin-expressing tumors. Results are from a representative experiment. (A, D, and E) Graphs show mean ± SEM with four or more mice per group. (F) Control (n = 5) or chemerin-expressing B16 (n = 5) was orthotopically implanted into C57BL/6 wild-type mice. Anti–Asialo GM1 depleting antibody was used to deplete NK cells in mice bearing chemerin-expressing tumors (NK depletion; n = 8). *, P < 0.05 comparing tumor size (mean ± SEM) in control or chemerin-expressing versus NK depletion by two-tailed Student’s t test. Data are representative of three experiments. (G) Chemerin-expressing B16 cells were orthotopically implanted in C57BL/6 wild-type and RAG1 KO mice (RAG KO; n = 9) and compared with control B16 cells implanted in WT mice. *, P < 0.05 comparing tumor size (mean ± SEM) in chemerin-expressing or RAG KO versus control by two-tailed Student’s t test. Data are representative of two experiments. (H and I) Control (H) or chemerin-expressing (I) B16 cells were implanted into WT or CMKLR1 KO mice (n = 5/group), and tumor growth was assessed. *, P < 0.05 comparing tumor size (mean ± SEM) in WT versus CMKLR1 KO with implanted chemerin-expressing tumors by two-tailed Student’s t test.

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