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. 2020 Aug 14;10:13838. doi: 10.1038/s41598-020-70788-3

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© The Author(s) 2020

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Granulocytes and T cells in BM and spleen comprise an immunosuppressive phenotype. (A) Spleen weight at day 40. Error bars represent SEM of biological repeats. n = 5–6. (B) Scheme of experimental design. Granulocytes (CD45⁺CD11b⁺Ly6C^intLy6G⁺) and T cells (CD45⁺CD3⁺) were analyzed and isolated. This scheme was designed by using graphical elements from BioRender. (C, D) Quantification of granulocytes and T cell population in the spleen of normal, 4T1-injected or 4T-Bone-injected mice. Error bars represent SEM of biological repeats. One way ANOVA was performed. n = 5–6. (E) Correlation between spleen weight and the abundance of granulocytes in the BM of 4T1 (n = 9) and 4T-Bone (n = 10) injected mice. (F) Correlation between Granulocytes and T cells in the BM of 4T1 (n = 9) and 4T-Bone (n = 10) injected mice. (E, F) Pearson correlation was performed. (G) qPCR analysis of the expression of immunosuppressive signature expressed by granulocytes isolated from the BM (G) and the spleen (H) of normal/4T1/4T-Bone-injected mice at day 40. (G, H) Error bars represent SEM of biological repeats. One-way ANOVA was performed. n = 5. (I) qPCR analysis of the expression of activation markers in T cells isolated from the BM (I) and the spleen (J) of normal/4T1/4T-Bone-injected mice at day 40. (I, J) Mann–Whitney test was perfomed. Error bars represent SEM of biological repeats. n = 5. (K, L) Arginase activity in sorted granulocytes from the BM (K) and spleen (L), One-way ANOVA was performed. Error bars represent SEM of biological repeats. n = 5.