Fig. 2.

5-Nonyloxytryptamine (5-NL) improves T cell anti-tumor immunity in vivo. A-E C57BL/6 J mice were subcutaneously injected with 5 × 10⁵ B16.GP33 cells. 7 days post-tumor injection, mice were randomized into two groups and treated daily with 6.25 mg/kg of 5-NL or with vehicle for five consecutive days. Mice were sacrificed on 13 days post tumor-inoculation. A Tumor sections were stained for CD8⁺ T cells using immunofluorescence (a representative image of n = 4 is shown, scale bar indicates 50 µm). B Numbers of tumor infiltrating CD8⁺ and CD4⁺ T cells, Treg’s (CD4⁺CD25⁺FOXP3⁺), monocytes (CD11b⁺Ly6C^highLy6G⁻), granulocytes (CD11b⁺Ly6G^highLy6C^low), tumor associated macrophages (TAMs, CD11b⁺F4/80^highLy6C^lowLy6G⁻) and dendritic cells (DCs, CD11c⁺MHC-II⁺) were assessed using flow cytometry (n = 6–10). C In addition to the tumor inoculation and 5-NL treatment described in (A), C57BL/6 J mice were also treated with a CD8⁺ T cell depleting antibody (anti-CD8) on days -2, -1 and 7 pre and post tumor cell inoculation. Tumor volume was measured (n = 4–8). D-E Tumor and tumor-draining lymph node infiltrating CD8⁺ T cell markers, intracellular GZMB as well as tetramer were assessed by flow cytometry from mice sacrificed at day 13 (upper panel) or day 20 (bottom panel) post tumor inoculation (n = 5–11). F C57BL/6 J mice were infected with 2 × 10⁵ pfu of LCMV Armstrong and treated daily with 6.25 mg/kg of 5-NL or vehicle for 5 consecutive days starting at day 1 post-infection. 10 days post-infection, cells from the blood, spleen and liver were re-stimulated with LCMV-specific gp33 epitope followed by staining for IFNγ using FACS analysis (n = 5). Tet-gp33⁺ CD8⁺ T cells in the blood, spleen and liver were measured 10 days post-infection (n = 5). Error bars indicate SEM; *P < 0.05 as determined by a Student´s t-test (unpaired, 2 tailed), or a two-way ANOVA with a Tukey’s post-hoc test