Extended Data Fig. 5: p38α-mediated IFNAR1 downregulation induces CXCL1/CXCL3/CXCL5-CXCR2 axis which is critical for the neutrophil recruitment.

a. Representative flow cytometry analysis of myeloid cell subpopulations (above) and the quantification of these cell subpopulations (below) in the lung tissues of WT and SA mice treated with SFM or B16F10 TCM. Quantitative data shown as mean±SEM (n=4 mice per group). Two-way ANOVA and Sidak’s multiple comparisons test were performed. b. Representative flow cytometry analysis of the purity of the isolated granulocytes from lung tissues of WT and SA mice treated with B16F10 TCM. Similar results were obtained from three independent experiments. c. Antigen-specific proliferation of CD8⁺ T cells in the presence of isolated granulocytes (Ly-6G⁺) from the lung tissues of WT and SA mice treated with B16F10 TCM at a ratio of 1:1, 1:2 or 1:4 measured as the uptake of ³H thymidine and presented relative to that in the absence of granulocytes which set as 100%. Splenic PMN-MDSC isolated from MC38 tumor-bearing mice served as a positive control for the suppression. Data shown as mean±SEM (n=6 mice for WT and SA group, n=3 mice for PMN-MDSC). Two-way ANOVA and Sidak’s multiple comparisons test were performed. d. qPCR analysis of mRNA levels of the indicated chemokines in WT lung fibroblasts 6 hr after SFM, conditioned media from normal lung fibroblasts (FCM), or TCM from different tumor cells including B16F1, B16F10, MH6499c4, and E0771. Data shown as mean±SEM (n=3 biologically independent samples). Two-tailed Unpaired t test was performed for the comparisons between two groups. e. qPCR analysis of mRNA levels of the indicated chemokines in the lung tissues of WT mice treated with SFM (n=3 mice), B16F1 TCM (n=4 mice) or B16F10 TCM (n=4 mice). Data shown as mean±SEM. Two-way ANOVA and Tukey’s multiple comparisons test were performed. f. Representative flow cytometry analysis (left) and the quantification of percent of neutrophils (right) in the lung tissues of WT mice treated with SFM (n=5 mice), B16F10 TCM plus vehicle (n=4 mice) or CXCR2 inhibitor (n=5 mice). Quantitative data shown as mean±SEM. Two-tailed Unpaired t test was performed for the comparisons between two groups. g. qPCR analysis of mRNA level of Cxcl1 in the lung tissues of SA mice intranasally administered with control (n=3 mice) or CXCL1 (n=7 mice) expressing adenovirus. Data shown as mean±SEM. Two-tailed Unpaired t test was performed for the comparison. h. The percent of total myeloid cells in the lung tissues of SA mice intranasally administered with control (n=4 mice) or CXCL1 (n=5 mice) expressing adenovirus. Data shown as mean±SEM. Two-tailed Unpaired t test was performed for the comparison.