Fig. 6

The timing by which LPS is administered relative to ACT therapy differentially impacts treatment outcome and regulates the innate and adaptive immune system. a Schematic showing the time at which LPS is administered to tumor bearing mice relative to the tripartite ACT treatment regimen. One day after TBI, mice received an ACT treatment comprised of the adoptive transfer of 5e⁵ cultured pmel-1 T cells, fowlpox hgp100 vaccination and hIL-2 or were left untreated. Either on day prior (b), during (c) or one day after (d) ACT, mice received 2 μg of LPS or were left untreated. Data shown (mean ± SEM, 5 mice/group) are representative of 4 independent experiments. 5Gy (black diamond) vs. 5Gy PFI (white diamond, **P < .01), 5Gy PFI pre-LPS (red circle, *P < .05), 5Gy LPS during PFI (blue circle, **P < .01) or 5Gy PFI post-LPS (green circle, **P < .01), ANOVA. 5Gy PFI (white diamond) vs. 5Gy PFI pre-LPS (red circle, *P < .05), 5Gy LPS during PFI (blue circle, NS) or 5Gy PFI post-LPS (green circle, **P < .01), ANOVA. (e–g) Splenocytes were harvested from irradiated mice on day 7 post-T cell infusion and absolute cell counts were determined by flow cytometry. *P < .05, **P < .01, unpaired t-test. (h & i) CD11c^hi dendritic cells were sorted by flow cytometry from single-cell B16F10 tumor suspensions prepared 6 days after ACT treatment from mice given LPS at different time points (as shown in scheme A). Tumor-infiltrating DCs were co-cultured for 4 days with CFSE-labeled, negatively-isolated pmel CD8⁺ T cells at a 10:1 T cells: APC ratio. DCs were exposed to antigen during in vivo fowlpox vaccination. Tumor antigen was not added to the co-culture. Representative flow cytometry plots (h) and dot plots (i) of CFSE dilution of pmel-1 cells. ****P < .0001, **P < .01, ANOVA