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Supplementary figures and tableFigure S1. Phosphoflow gating strategy for IFNγ and IL-6 signaling responsiveness. (a) Phosphoflow cytometry gating strategy for IFNγ-induced STAT1 phosphorylation in peripheral monocytes from BC patients. (b) The expression levels of basal phosphorylated STAT1 (pY701) in peripheral monocytes at diagnosis from relapsed (n=22), relapse-free (n=96) BC patients and healthy donors (HD). One-way ANOVA. **p<0.01. (c) expression levels of total intracellular STAT1 in peripheral monocytes at diagnosis from relapsed (n=8), relapse-free (n=12) BC patients, and healthy donors (n=12). One-way ANOVA. (d) frequencies of peripheral monocytes in PBMCs at diagnosis from relapse (n=22), relapse-free (n=96), and healthy donors (n=22). One-way ANOVA. (e) IFNγ signaling response in peripheral monocytes was compared between BC patients whose blood were collected at relapse (n=10) and patients who achieved and remained in remission for at least 3 years after their most recent relapse (n=10). Mann-Whitney test. *p<0.05.Figure S2. Phosphoflow gating strategy for IL-6 signaling responsiveness. Phosphoflow cytometry gating strategy for IL-6-induced STAT1/3 phosphorylation in peripheral na?ve CD4+ T cells.Figure S3. IFNγ signaling responsiveness not associated with levels of CXCR4, CCR2 or VEGFR2 on peripheral blood monocytes. (a-c) Cryopreserved PBMCs from BC patients were thawed and rested for 16hrs. PBMCs were stimulated with IFNγ at 50ng/ml for 15 minutes (n=20). IFNγ induced STAT1 phosphorylation (ΔMFI) and the levels of chemokine receptors CXCR4 (a), CCR2 (b) and VEGFR2 (c) in peripheral monocytes were examined by flow cytometry. The associations between IFNγ signaling response and levels of these chemokine receptors were examined by Spearman’s correlation coefficient test. ns=not significant. Figure S4. IFNγ signaling responsiveness in peripheral blood monocytes not associated with IFNγ plasma levels. (a) IFNγ plasma levels in relapse-free (n=96) and relapsed (n=22) BC patients at diagnosis. Mann-Whitney test. (b) The association between IFNγ plasma levels and IFNγ induced STAT1 phosphorylation in monocytes from the BC patients (n=80) was examined by Spearman’s correlation coefficient test. Figure S5. Correlation between Oncotype DX scores and IFNγ signaling response in peripheral monocytes. (a) correlation between Oncotype DX scores and the IFNγ-induced pSTAT1 in paired peripheral monocytes from BC patients at diagnosis (n=46, ER+HER2-). Spearman’s correlation coefficient test. (b) Oncotype DX score and IFNγ-induced pSTAT1 in paired peripheral monocytes from relapsed (n=8) and relapse-free (n=38) patients. Figure S6. IFNγ signaling responsiveness in non-classical monocytes from BC patients not correlated with clinical outcome. (a) Representative flow plots showing IFNγ-induced pSTAT1 in non-classical monocytes (CD16hiCD14-/lo). (b) IFNγ-induced pSTAT1 were compared between classical and non-classical monocytes from BC patients at diagnosis (n=40). Wilcoxon test. **p<0.01. (c-d) IFNγ-induced pSTAT1 in non-classical monocytes (c) and frequency of non-classical monocytes (% of CD33+ myeloid cells) (d) in relapse-free (n=33) and relapsed (n=7) BC patients. Figure S7. IFNγ signaling responsiveness does not correlate with M2-like protein expression in peripheral monocytes from BC patients. (a) Representative flow cytometry plots showing the expression levels of MRC1 and CD206 in peripheral monocytes. (b) correlation between IFNγ-induced pSTAT1 and MRC1 levels in peripheral monocytes from BC patients (n=12, ER+HER2-). Spearman’s correlation coefficient test. (c) Representative flow cytometry plots showing the expression levels of CD163 in peripheral monocytes. (d) correlation between IFNγ-induced pSTAT1 and CD163 levels in peripheral monocytes from BC patients (n=12, ER+HER2-). Spearman’s correlation coefficient test.Table S1. Univariate and multivariate analysis of IFNγ signaling responsefor RFS of BC patients (n=78)UnivariateMultivariate *Hazard ratio (high vs low ΔMFI)(95% CI)0.08(0.02-0.36)0.07(0.01-0.48)p-value0.0010.0007Note:* adjusting for age, tumor stage, grade, nodal status and subtype ................
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