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. 2020 Jan 31;11:660. doi: 10.1038/s41467-019-12901-3

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — Increased T cell expansion in the spleen and durable tumor infiltration with NKTR-214 treatment. a Time-course bioluminescence imaging (BLI) of firefly luciferase-labeled pmel-1 T cell trafficking in vivo. Representative images on days 5 and 14, five replicate mice per group. T cell expansion in spleen (upper panels, SIDE); mobilization and persistence in tumor (lower panels, FRONT). Scale bar shows the radiance, going from a minimum of 3.0 × 10⁵ e to a maximum of 5.0 × 10⁶ photons second⁻¹ cm⁻² steradian⁻¹. b, c Quantitative imaging analysis of region of interest (ROI) of spleen (b) and tumor (c) through day 23 after ACT of pmel-1 T cells expressing luciferase. Mice were treated with the combination of ACT and NKTR-214 (0.8 mg/kg, q9dx3, i.v.) or with IL-2 (0.4 mg/kg, qdX3 every 9 days for 3 cycles, i.p.), or vehicle control. Signal in spleen reached the peak at day 5 for ACT + NKTR-214 compared to ACT + IL-2. *p < 0.0001 ACT + NKTR-214 compared to ACT + IL-2, ^#p < 0.0001 compared to ACT + vehicle, two-way ANOVA with Tukey multiple comparisons test, n = 5, mean ± SEM. d Representative immuno-PET/CT images acquired on day 5 after treatment with ACT + IL-2 or ACT + NKTR-214 (n = 3/group) using Zr-89-labeled anti-CD8 cys-diabodies (cDb). Scale bar represents the percent-injected dose per gram of tissue (%ID/g) detected, from low (black) to high (red) intensity. e Ex vivo analysis of T cell biodistribution in organs harvested at 24 h post-injection of Zr-89 cDb. %ID/g: injected dose per gram. The diabody and residualizing radionucleotide undergo renal clearance, hence the high non-specific signal from kidneys. Mean ± s.e.m (n = 3),*p < 0.05, ***p < 0.001, ****p < 0.0001, unpaired t-test. f CD8+ staining of FFPE from spleen at day 5, representative image of three replicates per group. HALO software analysis showing intensity of marker expression, from low (blue) to high (red) expression level. g Digital pathology quantification of IHC slides using HALO software showing the percentage of CD8 T cell expansion in the spleen at different time points. ****p < 0.0001, mean ± s.e.m, unpaired t-test.