Fig. 4

CXCL1 recruits peripheral naive CD4⁺ T cells and induces their differentiation into Tregs in situ by activating the NF-κB/FOXP3 pathway. a The distributions of CD34⁺ blood vessels (red), CD62L⁺ naive T cells (yellow), and FOXP3⁺ Tregs (green) within the TME of mammary tumors were detected using the tissue immunofluorescence assay. Scale bar = 5 μm. b The expression levels of CXCR2 in naive CD4⁺ T cells sorted from the mammary tumors were detected by flow cytometry. c Flow cytometry assay indicated that 10–30 ng/ml CXCL1 treatment for 24 h significantly increased the chemotaxis efficacy of naive CD4⁺ T cells, whereas CXCL1/CXCR2 blockage by 2 μM SB225002 (CXCR2 inhibitor) partially abrogated that effect. d–e The expression levels of FOXP3 in 293 T cells (d) and naive CD4⁺ T cells (e) when treated as indicated for 24 h were detected by Western blot assay and immunofluorescence assay, respectively. Scale bar = 3 μm. f–g The mRNA expression level (f) and the promoter activity of FOXP3 gene in naive CD4⁺ T cells when treated as indicated for 24 h were detected by qPCR and double luciferase reporter gene assay, respectively. h–i Western blot assay (h) and cell immunofluorescence assay (i) indicated that CXCL1 treatment (10–30 ng/ml) for 24 h induced the expression and nuclear translocation of NF-κb subunit p65, while CXCL1/CXCR2 blockage partially abrogated that effect. Scale bar = 3 μm. j CHIP-PCR assay validated that CXCL1 treatment (10–30 ng/ml) for 24 h promoted the binding of NF-κb subunit p65 with FOXP3 promoter region, while 10 μM Bay11-7082 treatment (NF-κb inhibitor) partially abrogated that effect. N = 3. *p < 0.05; **p < 0.01