Fig. 1
A pharmacological screen identifies the serotonin agonist 5-Nonyloxytryptamine (5-NL) as potentiating T cell mediated anti-tumor immunity. A Screen schematic is shown. B-F Mice were infected with 2 × 105 pfu of LCMV-Armstrong. 14 days post infection, splenic pan-T cells were purified. B Splenic T cells were analyzed using FACS to obtain the ratio of CD8+, CD4+ and CD4+CD25+FoxP3+ T cells shown as percent composition (n = 3). C CD8+ T cells were evaluated for various surface markers using flow cytometry (n = 3). D Co-cultured LCMV-primed splenic pan-T cells and B16.GP33 cells were treated with 770 pharmacological compounds at a concentration of 1 μM. T cells were removed from co-culture at 16 h. Tumor-cell viability was assessed using the MTT assay 48 h post-treatment. Viability for each compound was expressed as a fraction relative to control (B16.GP33 cells + T cells). Potential hit compounds below the cut-off of 0.7 are shown in red. (E, Left Panel) B16.GP33 and B16 cells were co-incubated with LCMV-primed splenic pan-T cells with and without 5-NL (1 μM) as described in D and tumor cell viability was assessed using the MTT assay (n = 3–5). (E, Right Panel) B16.GP33 cells were co-incubated with LCMV-primed splenic pan-T cells with and without T-cells for 72 h and the IC50 was determined using the MTT assay (n = 4). F B16.GP33 and B16 murine melanoma cells were co-incubated with LCMV-primed splenic pan-T cells and 5-NL for 16 h. Intracellular staining of CD8+ T cells for TNFα, Granzyme B (GZMB) and IL-2 was measured using flow cytometry and normalized to its own respective cell line controls (n = 6). G C57BL/6 J or (H) NSG mice were subcutaneously injected with 5 × 105 B16.GP33 cells. 7 days post-tumor injection, mice were randomized and into two groups and treated daily with 6.25 mg/kg of 5-NL or vehicle for five consecutive days and tumor volume was measured (n = 5–12). Error bars indicate SEM; *P < 0.05 as determined by a Student´s t-test (unpaired, 2 tailed), one or two-way ANOVA with a Dunnett’s post-hoc test