Virus-specific CD8⁺ T cell activation by Lm-HCV-DC challenge with Tim-3 blockade.

<p>a) Tim-3 expression on virus-specific CD8⁺ T cells from HCV-infected and resolved individuals. M/M_Ø isolated from HCV-infected and HCV-resolved subjects were stimulated with GM-CSF and IL-4 for 5 days to induce iDC, which were infected with Lm-NS5B for 2 days to induce mDC, and then incubated with autologous T cells in the presence of IL-2 for 5 days to activate CTLs. The cells were immune-staining with APC-Tim-3, FITC-CD3, PerCP-CD8, and PE-HCV-NS5B-tetramer (HCV-Tet). Data are shown as representative dot plots of gating strategy for Tim-3 expression on HCV-Tet+CD3+CD8+ CTLs on the left, and mean ± SD from 10 HCV-infected versus 6 HCV-resolved subjects (2 spontaneously resolved and 4 HCV-treated SVR subjects) on the right; *P<0.05. b) HCV-specific CD8⁺ T cell activation by Lm-HCV-DC challenge concomitant with Tim-3 blockade. M/M_Ø isolated from HCV-infected patients were stimulated with GM-CSF and IL-4 for 5 days to induce iDC, which were infected with Lm-control or Lm-NS5B for 2 days to induce mDC, and then incubated with autologous T cells in the presence of IL-2 for 5 days to activate CTLs. The cultures were carried out in the presence of anti-Tim-3 or IgG antibodies during induction of iDC and mDC as well as activation of T cells. HCV-specific T cell activation was assessed by measuring the frequencies of HCV-NS5B-tetramer⁺ CTLs (HCV-Tet⁺) in CD3⁺CD8⁺ T cells. Data are shown as representative dot plots of the tetramer⁺ frequencies in the gated CD3⁺CD8⁺ T cells (left), and summary of mean ± SD from 6 HCV-NS5B tetramer⁺ patients; *P<0.05, **P<0.01.