Figure 1.
NK cell frequency and altered subset distribution in the periphery and intrahepatic compartment.
(A) Representative density plots gated on CD3- PBMC and co-stained for CD56 and CD16 to identify NK cells from a healthy control and a CHB patient. (B) Summary data of the proportions of CD56bright subset in the periphery of CHB patients with low ALT (n = 35, ALT <50IU/L, median 34) compared to high ALT (n = 29, median ALT 112) and healthy controls (n = 31). (C) Frequency of circulating NK cells in CHB patients with low ALT and high ALT and healthy controls. (D) Density plots of NK cells from peripheral blood and intrahepatic lymphocytes from a representative CHB patient. (E) Paired cumulative results of peripheral and intrahepatic CD56bright NK cells frequencies from 8 patients with CHB. (F) NK cell frequency in peripheral blood and intrahepatic compartment from 8 patients with CHB with paired samples. The non-parametric Mann-Whitney U test was used to compare data between groups and the Wilcoxon signed rank test was used between paired variables. *p<0.05 or ** p<0.01 designates values that differ significantly between groups. Ctr = healthy controls.
Table 1.
Characteristics of study population.
Figure 2.
Skewed NK cell effector function in CHB is only partially corrected during therapy.
(Panels A–C) Representative density plots from a healthy control and HBV patients with low ALT (ALT <50 IU/L, median 33) and raised ALT (ALT>50 IU/L, median 112) and summary data for TRAIL expression, IFN-γ production and CD107 expression. (D) Summary bar charts of CD56bright proportions, NK cell TRAIL expression and NK cell IFN-γ production from healthy, CHB and patients on antiviral therapy. Results are expressed as mean ± SEM. Rx = treated patients. *p<0.05, **p<0.01, ***p<0.001 by Mann-Whitney test.
Figure 3.
IL-10 is elevated in CHB and suppresses NK cell IFN-γ production.
(A) Levels of cytokines IL-17A and (B) IL-10 determined using Cytometric Bead Arrays flex sets using sera from 13 healthy controls, 14 low ALT (median ALT 35, all eAg-) and 21 high ALT patients (median ALT 115, 13eAg-). (C) Cumulative IL-10 results including therapy group (n = 13, median ALT 25). (D) Representative density plots of the effect of exogenous IL-10 on IFN-γ production by NK cells from a CHB patient and (E) paired cumulative results from 19 CHB patients. (F) Summary bar charts of the effect of exogenous IL-10 on the expression of TRAIL and CD107 in 5 CHB patients. Results are paired and expressed as mean ± SEM. Stimulus = IL12+IL18. Significance determined by the Mann-Whitney test for comparison between groups and the Wilcoxon signed rank test for paired data, *p<0.05, **p<0.01, ***p<0.001.
Figure 4.
IL-10 blockade alone or in combination with TGFβRII blocking restores NK cell IFN-γ production.
(A) Representative density plot from a CHB patient of peripheral NK cell IFN-γ production in the presence of anti-IL-10 and anti-IL10 receptor blocking mAb. (B) Paired summary data from CHB patients with either active disease (High ALT median 104, n = 13) or (C) inactive disease (Low ALT median 33, n = 9). (D) Fold change in IFN-γ produced by total NK cells following IL-10 blockade in both groups of patients. (E, F) Representative density dot plots from a CHB patient and summary bar chart of paired results from 11 patients (n = 11 median ALT 42) of NK cell IFN-γ production following IL-10 blockade alone or in combination with anti-TGFβRII blocking antibodies. Stimulus = IL12+IL18. Significance determined by the Mann-Whitney test for comparison between groups and the Wilcoxon signed rank test for paired data, *p<0.05, **p<0.01.
Figure 5.
Blockade of IL10/TGF enhances intrahepatic NK cell IFN-γ production.
(A) Representative density plots and (B) histograms for total intrahepatic NK cell and CD56bright subset IFN-γ production upon blockade with anti-lL-10, anti-IL10 receptor and anti-TGFβRII blocking antibodies. Paired summary bar charts of fold change increase in the percentage and mean fluorescence intensity (MFI) of NK total and CD56bright IFN-γ+ cells in the periphery and intrahepatic compartment of 7 CHB (median ALT 56). Results are expressed as mean ± SEM. Stimulus = IL12+IL18. *p<0.05 by Wilcoxon signed rank test.
Table 2.
Patient characteristics with available liver biopsy specimens.