Fig. 3.

The effect of FTY720 treatment on dynamics and distribution of CD8⁺ cells in response to anti–PD-1 (αPD-1) treatment. (A) MC38 tumor-bearing mice were treated with FTY720 twice a week, at 20 μg per mouse, starting either at day 0 (cohort i, n = 8) or day 7 (cohort ii, n = 5). The control cohort (cohort iii) did not receive FTY720 (n = 5). Mice were treated with αPD-1 and were subjected to PET-CT imaging according to the schedule shown in A (10⁵ MC38-GFP⁺ cells were injected at day 1 followed by a suboptimal dose of treatment to maximize the effect of FTY720 treatment; 100 μg αPD-1 antibody on days 8, 11, and 14). (B) Survival curves for each of the 3 cohorts. Mice were killed when tumors reached 2 cm in diameter or on ulceration. Log-rank statistical test was performed to determine significance between each cohort (*P < 0.05; **P < 0.01; ***P < 0.001). (C) PET-CT maximum intensity projection images of a B6 mouse, injected with αCD8 ⁸⁹Zr-PEG20-VHH, 11 d after inoculation of the MC38 tumor (box); for each of the cohorts that received FTY720 (C, i and ii) and the control cohort (C, iii), PET images of a cross-section of the tumors on days 11 and 20 are shown: (Top) day 11, (Bottom) day 20 after tumor inoculation. Tumors, as identified by CT, are marked by the outline. The PET signals in the tumor are rendered as a heat map. Below each image is the corresponding 3D rendition, in which the z axis represents the strength of the PET signal in arbitrary units.