Fig 2: CCL11 blockade inhibits the 4T1 cell-induced proportion of CD4+CD25+Foxp3+ Treg cells and the release of IL-2 and TGF-ß1 in vivo. BALB/c nude mice were intravenously injected with 4T1 cells and intraperitoneally injected with anti-CCL11 neutralizing (100 µg) every 48 h (n=6 per group). (A) Representative images of the histological inspection of mouse lungs at 21 days post-treatment. Scale bar, 100 µm. (B) Representative plots and (C) quantification of the proportion of CD4+CD25+Foxp3+ Treg cells, measured by flow cytometry. (D) Serum levels of CCL11, IL-2 and TGF-ß1, measured by ELISA. ****P<0.0001 vs. the control group; ####P<0.0001 vs. the 4T1 cell group. CCL11, C-C motif chemokine ligand 11; Foxp3, forkhead box P3; Treg, T regulatory; IL, interleukin; TGF, transforming growth factor.

Fig 3: Involvement of the STAT5 signaling pathway in the function of CCL11. CD4+ T cells collected from peripheral blood mononuclear cells of healthy individuals were treated with rhCCL11 (50 ng/ml) in the presence or absence of the STAT5 inhibitor CAS (1 µM) or vehicle (n=3 per group). (A) Representative plots and (B) quantification of the proportion of CD4+CD25+Foxp3+ Treg cells, measured by flow cytometry. (C) Western blots and (D) quantification of p-STAT5, STAT5 and Foxp3 protein expression levels, measured by western blotting. (E) IL-2 and TGF-ß1 levels in the cell media, measured by ELISA. ****P<0.0001 vs. the control group; ###P<0.001 and ####P<0.0001 vs. the rhCCL11 + vehicle group. CCL11, C-C motif chemokine ligand 11; rhCCL11, recombinant human CCL11; Foxp3, forkhead box P3; Treg, T regulatory; p, phosphorylated; IL, interleukin; TGF, transforming growth factor.

Fig 4: CCL11 blockade reduces the proportion of CD4+CD25+Foxp3+ Treg cells and the level of STAT5 activation in tumor-associated CD4+ T cells. CD4+ T cells collected from the peripheral blood mononuclear cells of patients with BC were incubated with anti-CCL11 neutralizing antibodies (0.1, 0.2, 0.5, 1 and 2 µg/ml; n=3 per group). (A) Proportion of CD4+CD25+Foxp3+ Treg cells, measured by flow cytometry. (B) CCL11, CCR3 and Foxp3 expression, measured by western blotting. CD4+ T cells were incubated with anti-CCL11 neutralizing antibody (0.2 and 2 µg/ml), and the expression levels of (C) p-STAT5 and STAT5, as well as (D) the serum content of IL-2 and TGF-ß1, were measured by western blotting and ELISA, respectively (n=3 per group). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 vs. the control group. CCL11, C-C motif chemokine ligand 11; Foxp3, forkhead box P3; Treg, T regulatory; BC, breast cancer; CCR3, C-C motif chemokine receptor 3; p, phosphorylated; IL, interleukin; TGF, transforming growth factor.

Fig 5: CCL11 is upregulated in patients with BC and is correlated with the proportion of CD4+CD25+Foxp3+ Treg cells. (A) Normalized CCL11 expression in TCGA BC tumors (n=1,041) and adjacent-normal breast tissues (n=112). (B) Serum content of CCL11 in patients with BC (n=100) and healthy individuals (n=50), measured by ELISA. (C) The CD4+CD25+Foxp3+ Treg cell proportion among CD4+ T cells from peripheral blood mononuclear cells of healthy individuals (n=10) and patients with BC (n=20), measured by flow cytometry. (D) Representative flow cytometry plots of the proportion of CD4+CD25+Foxp3+ Treg cells. (E) Correlation between the serum level of CCL11 and the proportion of CD4+CD25+Foxp3+ Treg cells in patients with BC (n=20). ****P<0.0001 vs. adjacent-normal or healthy controls. CCL11, C-C motif chemokine ligand 11; BC, breast cancer; Foxp3, forkhead box P3; Treg, T regulatory; TCGA, The Cancer Genome Atlas.