Membrane Bound CRT Fragment Accelerates Tumor Growth of Melanoma B16 Cell In Vivo through Promoting M2 Polarization via TLR4

Figure 4

tmCRT/39-272 promoted Treg differentiation through TGF-β secretion by M2 macrophages. (a) Female Foxp3-GFP C57BL/6 mice () were s.c. injected with B16-EGFP or B16-tmCRT/39-272 cells ( μL/mouse). Spleen cells were collected from the mice 12 days postinoculation. The proportions of Tregs were analyzed by FACS. FACS dot plots and the statistical results of Tregs are shown in panel. (b) Naïve CD4⁺ T cells purified from the spleens of Foxp3-GFP C57BL/6 mice, different doses (50%, 25%, 12.5%) of B16-EGFP or B16-tmCRT/39-272 cell supernatants were added to the wells for 72 h. Single cells were collected and the populations of Treg analyzed by FACS. (c) ELISA of TGF-β in the supernatants of C57BL/6 mouse bone marrow cells cultured in M-CSF conditioned medium, followed by the addition of γ-ray-treated B16-EGFP or B16-tmCRT/39-272 cells. (d) B16-EGFP or B16-tmCRT/39-272 cell supernatants (12.5%) were added to the naïve CD4⁺ T cells (purified from the spleens of Foxp3-GFP C57BL/6 mice) and treated with or without TGF-β-blocking antibody (10 ng/mL) for 72 h. Single cells were collected and analyzed by FACS. The experiments were repeated three times. , , , ns: not significant.

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