Ribavirin exerts differential effects on functions of Cd4+ Th1, Th2, and regulatory T cell clones in hepatitis C

Abstract

Ribavirin improves outcomes of therapy in chronic hepatitis C but its mode of action has still remained unclear. Since ribavirin has been proposed to modulate the host's T cell responses, we studied its direct effects on CD4(+) T cell clones with diverse functional polarization which had been generated from patients with chronic hepatitis C. We analysed in vitro proliferation ([(3)H] thymidine uptake) and cytokine responses (IL-10, IFN-gamma) at varying concentrations of ribavirin (0-10 µg/ml) in 8, 9 and 7 CD4(+) TH1, TH2 and regulatory T cell (Treg) clones, respectively. In co-culture experiments, we further determined effects of ribarivin on inhibition of TH1 and TH2 effector cells by Treg clones. All clones had been generated from peripheral blood of patients with chronic hepatitis C in the presence of HCV core protein. Ribavirin enhanced proliferation of T effector cells and increased production of IFN-gamma in TH1 clones, but had only little effect on IL-10 secretion in TH2 clones. However, ribavirin markedly inhibited IL-10 release in Treg clones in a dose dependent fashion. These Treg clones suppressed proliferation of T effector clones by their IL-10 secretion, and in co-culture assays ribavirin reversed Treg-mediated suppression of T effector cells. Our in vitro data suggest that--in addition to its immunostimulatory effects on TH1 cells--ribavirin can inhibit functions of HCV-specific Tregs and thus reverses Treg-mediated suppression of T effector cells in chronic hepatitis C.

Figure 1. Inhibition of TH cell proliferation by CD4⁺ Treg clones.

This figure illustrates that Treg clones generated from patients with chronic hepatitis C inhibit proliferation of autologous anti-CD3/anti-CD28 stimulated TH1 and TH2 clones. Inhibition of TH1 and TH2 clones was not altered when direct cell-contact was prevented by co-culture in a semipermeable transwell system. Co-culture experiments with a neutralising IL-10 antibody (10 µg/ml; clone 23738) prevented Treg-mediated suppression of TH1 and TH2 clones indicating that inhibition of TH1 and TH2 clones by Treg clones involved IL-10 production as a major soluble inhibitor. Proliferative responses were studied via [³H] thymidine assays. The figures present mean±SEM of experiments with TH1 (filled circles) and TH2 clones (open circles), respectively.

Figure 2. Effect of ribavirin on proliferation of differentially polarized CD4⁺ T cell clones.

Figure 2 shows dose response curves of proliferation ([³H] thymidine uptake, cpm = counts per minute; mean ± SEM) versus ribavirin concentrations (0, 1, 5 and 10 µg/ml) in anti-CD3/anti-CD28-activated Treg clones (A; n = 7), TH2 clones (B; n = 8) and TH1 clones (C; n = 7). Response curves were obtained by a non-linear curve fit with the GraphPad Prism Response Curve module (GraphPad Prism 4 Software, San Diego California, USA). Statistical significance was calculated by 1-way ANOVA with correction for repeated measurements.

Figure 3. Effect of ribavirin on cytokine secretion in differentially polarized CD4⁺ T cell clones.

Figure 3 illustrates dose response curves of cytokine release (pg/ml) versus ribavirin concentrations (0, 1, 5 and 10 µg/ml) in anti-CD3/anti-CD28-activated Treg clones (A; n = 7), TH2 clones (B; n = 9) and TH1 clones (C; n = 8). Dose response curves were calculated analogous to figure 2.

Figure 4. Influence of ribavirin on the proliferation of Treg clones in the presence of IL-2.

Figure 4 demonstrates that anti-CD3/anti-CD28-stimulated Tregs markedly proliferate in the presence of high amounts of recombinant IL-2 (100 IU/ml). Adding ribavirin at various concentrations (0, 1, 5 10 µg/ml) proliferation of Tregs was significantly reduced under these experimental conditions.

Figure 5. Reversal of Treg-mediated inhibition of T effector cell proliferation by ribavirin.

Figure 4A illustrates a representative co-culture experiment of TH1 clone #18 and Treg clone #4 in the presence of varied concentrations of ribavirin (0–5 µg/ml). Figure 4B provides a summary of all co-culture experiments of TH1 and TH2 effector clones with autologous Treg clones in the presence of varied concentrations of ribavirin (0–5 µg/ml). Each plot summarizes the effects for a defined concentration of ribavirin. The x-coordinate of each plot indicates proliferation (cpm = counts per minute) of a T effector clone without Tregs. The coordinate on the left y-axis shows the corresponding proliferation in the presence of autologous Treg clones (1∶1 ratio). Dots below the dotted line illustrate Treg-mediated inhibition of T effector cell proliferation. The straight line represents the linear regression line of all co-culture experiments. Intersection of this line with the right y-axis shows overall percent inhibition and its 95% confidence interval.