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. Author manuscript; available in PMC: 2013 Jul 15.
Published in final edited form as: J Immunol. 2012 Jun 15;189(2):755–766. doi: 10.4049/jimmunol.1200162

Fig. 6. Tim-3 blockade differentially promotes proliferation of Foxp3+ Tregs and Foxp3− Teffs through improving the phosphorylation of STAT-5 in CD4+CD25+ T cells from HCV patients.

Fig. 6

A) Tim-3 blockade on CD4+CD25+ T cells enhances Foxp3+ Treg expansion. Left panel, single representative dot plots of CFSE-labeled CD4+CD25+ T cells from HCV-infected patients that were pretreated with a control IgG or anti-Tim-3 overnight, then stimulated without or with anti-CD3/CD28 (1 µg/ml) and IL-2 (50U/ml) for 6 d; the cells were double-stained with Foxp3 and CFSE dilutions in Foxp3+ Tregs are shown. Right panel, summary data of proliferation of CD4+CD25+Foxp3+ Tregs from 8 HCV-infected patients under various ex vivo treatments. *P<0.05, **P<0.01. B) Tim-3 blockade improves the phosphorylation of STAT-5 in CD4+CD25+ T cells from HCV-infected patients. The purified CD4+CD25+ T cells from HCV-infected individuals were pretreated with a control IgG or anti-Tim-3 antibody overnight and then stimulated with anti-CD3/CD28 (1 µg/ml) and IL-2 (50U/ml) for 6 h, followed by Western blot to detect pSTAT-5 and total STAT-5. Densitometry data of phosphorylated STAT-5, normalized by total STAT-5 as loading control, from 3 HCV-infected patients is shown below. **P<0.01. C) Tim-3 blockade on CD4+ T cells ex vivo promotes expansion of Foxp3− Teffs more substantially, thus correcting the imbalance of Foxp3+ Tregs/Foxp3− Teffs established during chronic HCV infection. Purified CD4+ T cells were treated as described in A), and summary data of the Foxp3+ Tregs/Foxp3− Teffs ratio changes under various treatments using purified CD4+ T cells from 8 HCV-infected patients is shown. **P<0.01. D) Tim-3 blockade on CD4+ T cells reduces the ratio of CD25+Foxp3+ Tregs in the CD4+ T cells from HS and HCV-infected individuals. The purified CD4+ T cells were pretreated with a control IgG or anti-Tim-3 overnight, and then stimulated with anti-CD3/CD28 (1 µg/ml) for 48 h, followed by flow cytometric analysis of Foxp3 expression in CD25+ T cells. Summary data of CD25+Foxp3+ Tregs in the purified CD4+ T cells under various treatments from 8 HS and 8 HCV-infected patients is shown in the right panel. **P<0.01.