Fig 2: The non-transcriptional activity of HIF-1a is necessary for IL-15/IL-15Ra complex-induced reduction of CX3CL1 expression in tumor cells. (A) Western blotting to detect STAT3, p-STAT3, STAT5 and p-STAT5 expression in 4T1 tumor cells stimulated by IL-15Rc or GMCSF. (B) CX3CL1 mRNA levels in 4T1 tumor cells alone or cocultured with Raw264.7 cells in the presence of IL-15 or not were assessed by RT-qPCR. (C) CX3CL1 mRNA levels in 4T1 cells cultured in normoxic and hypoxia conditions were assessed by RT-qPCR. (D) Western blotting to detect CX3CL1 and HIF-1a expression in 4T1 tumor cells cultured in normoxic or hypoxia conditions. (E) Western blotting to detect CX3CL1 and HIF-1a expression in 4T1 tumor cells alone or cocultured with Raw264.7 cells in the presence of IL-15 or not in normoxic or hypoxia conditions. (F) Western blotting to detect CX3CL1 and HIF-1a expression in 4T1 breast tumors on days 7, 14, 21, and 28 after inoculation. (G) Correlation analysis between CX3CL1 and HIF-1a expression in 4T1 breast tumors. R2 values by linear regression. (H-I) Western blotting and RT-qPCR to detect CX3CL1 expression in HIF-1a-OE and HIF-1a-KO 4T1 tumor cells. (J) Western blotting to detect CX3CL1 expression in HIF-1a-KO 4T1 cells stimulated by different culture mediums from BMDMs. (K) CHX-chase analyses of CX3CL1 degradation in control, HIF-1a-OE, or HIF-1a-KO 4T1 tumor cells. Cells were incubated with CHX and were harvested at the indicated time points. Whole-protein lysates were harvested and ran for Western blots using the anti-CX3CL1 antibody. Half-lives of CX3CL1 proteins in control, HIF-1a-OE, or HIF-1a-KO 4T1 tumor cells. (L) The control and HIF1-1a-KO 4T1 cell lysate was immunoprecipitated with anti-CX3CL1 antibodies and immunoblotted with anti-ubiquitin antibodies. Abbreviations: CHX, cycloheximide.

Fig 3: GMSCF induces the expression of ?c to regulate tumor cells’ response to the IL-15/IL-15Ra complex. (A) Western blotting to detect CX3CL1 in 4T1 cells stimulated by the IL-15/IL-15Ra complex (IL-15Rc) or not. (B) Flow cytometric analysis of IL-2R/IL-15Rß and ?c in 4T1 tumor cells cocultured with Raw264.7 cells. (C) Western blotting to detect CX3CL1 expression in 4T1 tumor cells cocultured with Raw264.7 cells in the presence of IL-15 or neutralizing antibodies against IL-2R/IL-15Rß and ?c. (D) Flow cytometric analysis of ?c in 4T1 tumor cells overexpressing ?c. (E) Western blotting to detect CX3CL1 expression in ?c-OE 4T1 tumor cells stimulated by IL-15Rc. (F) GMCSF mRNA levels in Raw264.7 cells alone or cocultured with 4T1 cells (n = 3). (G) GMCSF mRNA levels in 4T1 tumor cells alone or cocultured with Raw264.7 cells (n = 3). (H) Flow cytometric analysis of IL-2R/IL-15Rß and ?c in 4T1 tumor cells treated with GMCSF. (I) Western blotting to detect CX3CL1 expression in 4T1 tumor cells stimulated by IL-15Rc or GMCSF. (J) Western blotting to detect CX3CL1 expression in 4T1 tumor cells stimulated by different culture mediums of BMDMs or TCM-induced BMDMs treated with IL-15 or neutralizing antibodies against GMCSF (anti-GMCSF). All values are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, by Student's t-test. Abbreviations: ?c, ? chain; TCM, tumor conditioned medium; GMCSF, granulocyte-macrophage colony-stimulating factor.

Fig 4: IL-15Ra+ TAMs release the IL-15/IL-15Ra complex to decrease chemokine CX3CL1 expression in tumor cells. (A-B) Flow cytometric analysis and quantification of Ki-67+CD8+ T cells in tumors coinjected with control iBMDMs or IL-15Ra-KO iBMDMs. (C) IL-15Ra+ TAMs and IL-15Ra– TAMs were sorted and cocultured with CD8+ T cells. Representative CFSE proliferation profiles for CD8+ T cells are shown. (D) Correlation analysis of T cell-related chemokine genes and CD4, CD8A in various tumors (breast, colon, kidney, lung, ovarian, uterine carcinomas). The ratio of the correlation coefficient of CD8A or CD4 with the same chemokine was further analyzed. Data were from the Expression Project for Oncology (ExpO) (http://www.intgen.org/, GEO GSE2109). The white boxes indicate that the ratio values were not within the setting interval of 0-2. (E) The mRNA expression of T cell-related chemokines in tumors coinjected with control iBMDMs or IL-15Ra-KO iBMDMs. (F) Western blotting to detect CX3CL1 expression in tumors coinjected with control iBMDMs or IL-15Ra-KO iBMDMs. (G) Representative IHC pictures of tumors coinjected with control iBMDMs or IL-15Ra-KO iBMDMs for detecting CX3CL1 expression. (H) Western blotting to detect CX3CL1 expression in Raw264.7 and 4T1 cells after cocultured in the presence of IL-15 or not. (I-K) Control or CX3CL1-KO 4T1 tumor cells were injected with admixed control iBMDMs or IL-15Ra-KO iBMDMs into Balb/c mice. Tumors were resected and measured 28 days later (n = 6). (L-M) Flow cytometric analysis of CD8+ T cells and CD3+CD8– T cells (pre-gate described in Supplementary Figure S2) in 4T1 breast tumors. The percentages of CD8+ T cells and CD3+CD8– T cells in total cells were quantified. (N) ELISA assay assessed IL-15Rc concentration in the coculture system. (O) ELISA assay to assess IL-15Rc concentration in the culture medium of control Raw264.7 cells and IL-15Ra-OE Raw264.7 cells stimulated by IL-15. (P) ELISA assay assessed IL-15Rc concentration in the coculture system in the presence of MMP inhibitor GM6001. (Q) ELISA assay to assess IL-15Rc concentration in the culture medium of control Raw264.7 cells and IL-15Ra-OE Raw264.7 cells stimulated by IL-15 in the presence of GM6001. (R) Western blotting to detect CX3CL1 expression in 4T1 tumor cells stimulated by different culture medium from BMDMs pre-treated with GM6001. (S) Western blotting to detect CX3CL1 expression in 4T1 tumor cells cocultured with Raw264.7 cells in the presence of GM6001. All values are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and not significant (ns) by Student's t-test. Abbreviations: IL-15Rc, IL-15/IL-15Ra complex; IL-15Ra-OE, IL-15Ra overexpression