Galectin-9 knockdown limits Treg-mediated inhibition of IFN-γ and IL-21 production by HCV-specific CD4 T cells.

<p>(<b>A</b>) Representative combined CFSE/ICS assay demonstrating reduced Treg-mediated inhibition upon knockdown of Gal-9 by siRNA. Plots depict production of IFN-γ, IL-21 and TNF-α by HCV-specific proliferating (CFSE^low) T cells in response to stimulation with recombinant HCV NS4 protein. PBMC cultures depleted of Tregs (Top panel) or with addition of Tregs (ratio 1∶4, Tregs∶CD25-depleted PBMCs) treated with either control siRNA (Middle panel) or <i>LGALS9</i> siRNA were stimulated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003422#s4" target="_blank">Materials and Methods</a>. Plots are gated on CD3⁺ CD8^neg lymphocytes. (<b>B</b>) Percentage of Treg-mediated inhibition of IFN-γ and IL-21 production by HCV-specific T cells upon stimulation with recombinant HCV proteins (NS3: black symbols or NS4: grey symbols) in the presence of Treg cells treated with either control or <i>LGALS9</i> siRNA, as described in (A) and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003422#s4" target="_blank">Materials and Methods</a>. The percentage inhibition of cytokine production upon addition of Tregs to the stimulated PBMC cultures was calculated using the following formula: (% CFSE^lowcytokine⁺CD8^neg T cells in absence of Tregs – % CFSE^lowcytokine⁺CD8^neg T cells in presence of Tregs)/% CFSE^lowcytokine⁺CD8^neg T cells in absence of Tregs×100. P-values were calculated using Wilcoxon signed rank test.