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. 2022 Jul 12;13:4032. doi: 10.1038/s41467-022-31551-6

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© The Author(s) 2022

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. 6 — a, b Flow cytometry analysis of percentages of CD3⁺CD4⁺ Th cells (a), and CD3⁺CD8⁺ CTLs (b) in blood of mice (n = 4) at 30 day after systemic administrations of saline, SPIN₀, SPIN_D2 (0.2 mL, 1.2 mg/mL) or free-drug mixture (8 mg/kg body weight for NLG919 and aPD-L1) with or without US irradiation (1.0 MHz, 1.2 W/cm², 50% duty cycle, 10 min). Saline – US versus drug − US: P = 0.0023; saline − US versus drug + US: P = 0.0006; drug + US versus SPIN_D2 + US: P = 0.0071 for CD3⁺CD4⁺ Th cells (a); saline − US versus drug − US: P = 0.0004; saline − US versus drug + US: P = 0.0001; drug + US versus SPIN_D2 + US: P = 0.0093 for CD3⁺CD8⁺ CTLs (b). c, d Flow cytometry analysis of percentages of CD3⁺CD4⁺ Th cells (c), and CD3⁺CD8⁺ CTLs (d) in spleen of mice (n = 4) after different treatments for 30 days. Saline − US versus drug − US: P = 0.0008; saline − US versus drug + US: P = 0.0005; drug + US versus SPIN_D2 + US: P = 0.0015 for CD3⁺CD4⁺ Th cells (c); saline − US versus drug − US: P = 0.0001; saline − US versus drug + US: P = 0.0002; drug + US versus SPIN_D2 + US: P = 0.0049 for CD3⁺CD8⁺ CTLs (d). e Representative H&E staining images of liver after 30 days of treatments in different groups (white arrows indicate the infiltrated lymphocytes). The experiments were repeated independently three times with similar results. f Heatmap to show relative fold of cytokine levels in serum of mice after different treatments for 30 days relative to those in saline control group. g, h Serum levels of ALT (g) and AST (h) in mice (n = 5) after different treatments for 30 days. Saline − US versus drug − US: P = 0.0010; saline − US versus drug + US: P = 0.0020; drug + US versus SPIN_D2 + US: P = 0.0054 for ALT (g); saline − US versus drug − US: P = 0.0001; saline − US versus drug + US: P < 0.0001; drug + US versus SPIN_D2 + US: P = 0.0013 for AST (h). i Summary comparison of the antitumor immunity and irAEs between SPIN_D2-mediated sono-immunotherapy and free-drug treatment. Data are presented as mean values ± SD. Statistical significance was calculated via two-tailed Student’s t test; **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.