Fig. 5. Foxp3⁺ Tregs are resistant, while IL-2-producing Foxp3⁻ Teffs sensitive to, TCR activation-mediated apoptosis that is reversible by Tim-3 blockade.

A) Dose- and cell-dependent apoptosis induced by TCR stimulation. PBMCs from HCV-infected patients were stimulated with anti-CD3 and anti-CD28 at 0, 2, and 5 µg/ml of each for 48 h, stained with FITC-CD4, APC-CD25, PerCP-Cy5.5-Foxp3, and PE-Annexin-V, followed by flow cytometric analysis. The cells were gated on CD4⁺CD25⁺ T cell populations and then Foxp3⁺ Tregs (upper panel) and Foxp3⁻ Teffs (lower panel), based on the isotype controls. The frequency of Annexin-V⁺ cells in the gated CD4⁺CD25⁺Foxp3⁺ Tregs or CD4⁺CD25⁺Foxp3⁻ Teffs is indicated in the right upper corner. B) Representative dot blots of Annexin-V expression on CD4⁺CD25⁺FoxP3⁺ Tregs (upper panel) and CD4⁺CD25⁺FoxP3⁻ Teffs (lower panel) by stimulation of purified CD4⁺CD25⁺ T cells from chronic HCV patients with varying doses of anti-CD3/CD28, followed by flow cytometric analysis of apoptosis as described above. The data are reproducible using purified cells from multiple HS and HCV. C) Tim-3 blockade lead to a decrease of apoptosis of purified CD4⁺CD25⁺ T cells from HCV patients. Purified CD4⁺CD25⁺ T cells from HCV-infected patients were pretreated with anti-Tim-3 or control IgG overnight, and then stimulated with anti-CD3/CD28 (2 µg/ml) for 48 h, followed by flow cytometric analysis of Annexin-V expression on CD4⁺CD25⁺FoxP3⁺ Tregs (upper panel) and CD4⁺CD25⁺ FoxP3⁻ Teffs (lower panel). Summary data obtained from 8 HCV-infected patients are shown in the right panel. **P<0.01. D) Tim-3 blockade reduced apoptosis of IL-2-producing Foxp3⁻ Teffs. Purified CD4⁺CD25⁺ T cells from HCV-infected patients were pretreated with anti-Tim-3 or control IgG overnight and then stimulated with anti-CD3/CD28 (2 µg/ml) for 48 h, followed by immunostaining and flow cytometric analysis of Annexin-V expression on IL-2⁻ Foxp3⁺ Tregs (upper panel) and IL-2⁺ FoxP3⁻ Teffs (lower panel). Summary data collected from 8 HCV-infected patients are shown in the right panel. **P<0.01.