Diminished virus-specific immunity induced by Lm-HCV-infected DCs negatively correlates with Tim-3 expression.

<p>a) Lm-based DC-targeting HCV vaccine only elicits a weak CTL response using immune cells from chronically HCV-infected individuals. M/M_Φ isolated from HCV-infected as well as HCV-resolved individuals were induced by GM-CSF and IL-4 for 5 days to iDC, which were subsequently infected by Lm-NS5B or Lm-control for 2 additional days to mDC; and then incubated with autologous T cells in the presence of IL-2 for another 5 days to generate CTL. Activation of the virus-specific CTLs was assessed by HCV-NS5B tetramer staining of CD3⁺CD8⁺ T cell frequencies as described in the Methods. Data are shown as representative dot plots of the staining with percentages of cells in each quadrant (left), and the mean ± SD of the percentages of HCV-tetramer⁺ CTLs (HCV-Tet⁺) in CD3⁺CD8⁺ T cells challenged by Lm-control or Lm-NS5B-infected DCs from 6 HCV-resolved (2 spontaneously resolved and 4 HCV-treated SVR subjects) and 8 HCV-infected individuals (upper right); *P<0.05, **P<0.01. b) Tim-3 expression on M/M_Φ, iDC, and mDC induced from HCV-infected and uninfected individuals. M/M_Φ were isolated from HCV-infected and uninfected individuals, iDC were induced by stimulation with GM-CSF/IL-4 for 5 days, and mDC were induced by Lm-infection for 2 days. Tim-3 expressions on M/M_Φ, iDC, and mDC were carried out as described in the Methods. Isotype-matched control antibodies and fluorescence minus one (FMO) controls were used to determine background levels of staining and adjust multicolor compensation as gating strategy. Data are shown as representative dot plots with percentages of cells in the gated area (left), and summary with mean of the percentages of Tim-3⁺ cells in M/M_Φ, iDC, and mDC from 14 HCV-resolved (4 spontaneously resolved, 4 HCV-treated SVR subjects, 6 HS) and 12 HCV-infected individuals (middle right). The relationship between Tim-3 expression on mDC and percentage of HCV-specific tetramer staining on CD3⁺CD8⁺ CTLs was analyzed by Pearson correlation (lower right). Each symbol represents one subject, and different individuals were shown by different symbols (^O for HS, ^Δ for SR, ^□ for SVR, and ^• for HCV-infected subject); *P<0.05, **P<0.01.