Fig 2: L-XRT hampers tumor growth, activates T cells, increases NK cells and M1 macrophages, and downregulates the inhibitory cytokine TGF-ß. (A), The one-tumor mouse model was established by injecting 344SQ-P cells (0.5 × 106) into the right hind legs of 129Sv/Ev mice (n=5/group), after which L-XRT (1 Gy × 2 fractions) was delivered when tumors reached around 7–8 mm in diameter. Anti-PD1 (200 µg/inj.) and anti-CTLA-4 (50 µg/inj.) were administered intraperitoneally on days 5, 8, 12, and 16 post 344SQ-P injection. Mice were euthanized when tumor reached 14 mm, and survival was plotted by the Kaplan-Meier method. Experiment was repeated twice, and similar patterns were detected. (B and C) dLNs from the L-XRT dose groups were harvested at 3 days after radiation and analyzed with flow cytometry. Both CD4 (No XRT vs 1 Gy × 2, p=0.0318) and CD8 cells (No XRT vs 1 Gy × 2, p=0.0001) were activated after two 1 Gy fractions, as depicted by the activation marker CD44. (D and E), spleens (spl), dLNs, and TILs were harvested at 48 hours after two 1 Gy fractions for phenotyping by flow cytometry. (D) Cells were gated on the CD45+ population and then on CD49b+ to identify NK cells (TILs no XRT vs TILs 1 Gy × 2, p=0.0328). (E) Cells were gated on CD45+ and then on Gr1intermediate and CD11b+ to identify macrophages, and further gated on F4/80+ CD38hi to identify M1 macrophages (No XRT vs 1 Gy × 2, p=0.0463). (F) NanoString molecular counts (nCounter immunology panel) showed significant reduction in local TGF-ß expression 24 hours after two 1 Gy fractions of L-XRT (p=0.0007). Data for each group was represented as mean±SD and Student’s t-tests were used to compare groups. *p=0.05 was considered to indicate statistical significance. dLNs, draining lymph nodes; L-XRT, low-dose radiation; PD1, programmed cell death protein 1; TGF-ß, tumor growth factor beta; TILs, tumor-infiltrating leukocytes.

Fig 3: Blocking NK-cell recruitment at the draining lymph node does not interfere with the generation of effector NK cells Absolute numbers of NK cells in one of the four draining lymph nodes in mice treated or not with the anti-CD62L antibody to inhibit NK-cell ingress in the lymph node from blood. Where indicated, mice were challenged with LPS.Absolute numbers of IFN-?+ NK cells in one of the four draining lymph nodes before and after LPS administration in mice in which NK-cell entry in the lymph node was blocked or not by anti-CD62L treatment.(Left panel) Absolute numbers of endogenous and adoptively transferred (CFSE+) NK cells in the draining and contralateral lymph node 5 h after LPS administration; (right panel) percent of endogenous and adoptively transferred NK cells on total lymphocytes in the draining and contralateral lymph node 5 h after LPS administration.Flow cytometry analysis of LPS-draining and contralateral lymph node from mice that received CFSE-labeled cells at the moment of LPS administration. Lymph node cells were stained with anti-CD3, anti-DX5, and anti-IFN-? antibodies, and the analysis was performed on gated CD3-DX5high cells. One representative of four independent experiments is shown.Tumor volume in mice treated or not with LPS; where indicated, mice received anti-CD62L antibody 12 h before LPS treatment.Percent of IFN-?+ NK cells within the tumor in mice treated or not with LPS and with the anti-CD62L antibody.Data information: (A, B, C, E, F) n (number of animals per group) = 4. Error bars depict SEM. Statistical significance was determined with a two-tailed t-test. NT, untreated.

Fig 4: GSK2857916 enhances intratumor immune cell infiltration and activation. Quantification by flow cytometry of immune cells infiltrating C57BL/6 mice bearing EL4-hBCMA tumors following treatment with GSK2857916 or isotype control (at 15 mg/kg), or saline vehicle. A, CD8+, CD4+, and regulatory (CD4+CD25+FoxP3+) T cells, and DC (CD11c+). B, T-cell activation markers CD25, CD69, and ICOS and the exhaustion marker PD-1. C, Quantification of the DC costimulatory molecule CD86, T-cell proliferation (Ki67) and cytotoxicity (granzyme B, GrzB) in TILs and NK cells (CD49b+). A one-way ANOVA statistical analysis was performed. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P = 0.0001. D, IHC for CD8 reactive cells in mice bearing EL4-hBCMA tumors treated with GSK2857914 (top) or GSK2857916 (bottom) at 15 mg/kg (as in Fig. 4). Representative images, 20× magnification. E, Quantification of calreticulin cell surface expression (MFI) on tumor cells in mice. F, Heatmap depicting a type I IFN-related gene signature following treatment with saline, isotype control or GSK2857916 (at 15 mg/kg) from the same experiment as in (A–C, E). Each column represents an individual mouse and each row an individual gene.