Dysfunctional B-cell activation in cirrhosis resulting from hepatitis C infection associated with disappearance of CD27-positive B-cell population

Abstract

Chronic hepatitis C virus (HCV) infection is a leading cause of cirrhosis and hepatocellular carcinoma (HCC). Both advanced solid tumors and HCV have previously been associated with memory B-cell dysfunction. In this study, we sought to dissect the effect of viral infection, cirrhosis, and liver cancer on memory B-cell frequency and function in the spectrum of HCV disease. Peripheral blood from healthy donors, HCV-infected patients with F1-F2 liver fibrosis, HCV-infected patients with cirrhosis, patients with HCV-related HCC, and non-HCV-infected cirrhotics were assessed for B-cell phenotype by flow cytometry. Isolated B cells were stimulated with anti-cluster of differentiation (CD)40 antibodies and Toll-like receptor (TLR)9 agonist for assessment of costimulation marker expression, cytokine production, immunoglobulin (Ig) production, and CD4(+) T-cell allostimulatory capacity. CD27(+) memory B cells and, more specifically, CD27(+) IgM(+) B cells were markedly less frequent in cirrhotic patients independent of HCV infection. Circulating B cells in cirrhotics were hyporesponsive to CD40/TLR9 activation, as characterized by CD70 up-regulation, tumor necrosis factor beta secretion, IgG production, and T-cell allostimulation. Last, blockade of TLR4 and TLR9 signaling abrogated the activation of healthy donor B cells by cirrhotic plasma, suggesting a role for bacterial translocation in driving B-cell changes in cirrhosis.

Conclusion: Profound abnormalities in B-cell phenotype and function occur in cirrhosis independent of HCV infection. These B-cell defects may explain, in part, the vaccine hyporesponsiveness and susceptibility to bacterial infection in this population.

Copyright © 2011 American Association for the Study of Liver Diseases.

Figures

Figure 1. Reduction of CD27+ memory B-cells in cirrhosis

A. Gating strategy for identification of CD19+ B-cells. B. Representative histograms of CD27 expression of CD19+ B-cells in healthy donors (HD), HCV with F1–F2 (early) fibrosis (EF), HCV cirrhotics (CIR) and HCV cirrhotics with hepatocellular carcinoma (HCC). C. Relative and absolute number of CD19+ B-cells in HD, HCV, EF, CIR and HCC.D. Relative and absolute number of CD27+ B-cells in HD, HCV, EF, CIR and HCC. E. Frequency of CD27+CD19+ B-cells in non-HCV cirrhotic patients relative to other patient groups. F. Distribution of CD27+IgM+ and CD27+IgG+ B-cells across patient groups. All statistical comparisons made using Kruskal-Wallis and pairwise Wilcoxon Rank Sum tests.

Figure 1. Reduction of CD27+ memory B-cells in cirrhosis

A. Gating strategy for identification of CD19+ B-cells. B. Representative histograms of CD27 expression of CD19+ B-cells in healthy donors (HD), HCV with F1–F2 (early) fibrosis (EF), HCV cirrhotics (CIR) and HCV cirrhotics with hepatocellular carcinoma (HCC). C. Relative and absolute number of CD19+ B-cells in HD, HCV, EF, CIR and HCC.D. Relative and absolute number of CD27+ B-cells in HD, HCV, EF, CIR and HCC. E. Frequency of CD27+CD19+ B-cells in non-HCV cirrhotic patients relative to other patient groups. F. Distribution of CD27+IgM+ and CD27+IgG+ B-cells across patient groups. All statistical comparisons made using Kruskal-Wallis and pairwise Wilcoxon Rank Sum tests.

Figure 1. Reduction of CD27+ memory B-cells in cirrhosis

A. Gating strategy for identification of CD19+ B-cells. B. Representative histograms of CD27 expression of CD19+ B-cells in healthy donors (HD), HCV with F1–F2 (early) fibrosis (EF), HCV cirrhotics (CIR) and HCV cirrhotics with hepatocellular carcinoma (HCC). C. Relative and absolute number of CD19+ B-cells in HD, HCV, EF, CIR and HCC.D. Relative and absolute number of CD27+ B-cells in HD, HCV, EF, CIR and HCC. E. Frequency of CD27+CD19+ B-cells in non-HCV cirrhotic patients relative to other patient groups. F. Distribution of CD27+IgM+ and CD27+IgG+ B-cells across patient groups. All statistical comparisons made using Kruskal-Wallis and pairwise Wilcoxon Rank Sum tests.

Figure 2. Correlation of CD27+ B-cell frequency and hepatic dysfunction

Spearman Rank correlation of CD27+ memory B-cell frequency and A. total bilirubin, B. serum albumin, C. platelet count, and D. international normalized ratio (INR).

Figure 3. Impaired activation of cirrhotic B-cells by CD40/TLR9 ligation

Difference in geometric mean fluorescence intensity of A. HLA-DR, B. CD86, C. CD40, and D. and frequency of CD70+ B-cells in HD, EF and CIR/HCC patients compared by Kruskal-Wallis and pairwise Wilcoxon Rank Sum tests. E. Spearman rank correlation between upregulation of CD70 expression upon CD40/TLR9 stimulation and baseline CD27+ B-cell frequency.

Figure 3. Impaired activation of cirrhotic B-cells by CD40/TLR9 ligation

Difference in geometric mean fluorescence intensity of A. HLA-DR, B. CD86, C. CD40, and D. and frequency of CD70+ B-cells in HD, EF and CIR/HCC patients compared by Kruskal-Wallis and pairwise Wilcoxon Rank Sum tests. E. Spearman rank correlation between upregulation of CD70 expression upon CD40/TLR9 stimulation and baseline CD27+ B-cell frequency.

Figure 3. Impaired activation of cirrhotic B-cells by CD40/TLR9 ligation

Difference in geometric mean fluorescence intensity of A. HLA-DR, B. CD86, C. CD40, and D. and frequency of CD70+ B-cells in HD, EF and CIR/HCC patients compared by Kruskal-Wallis and pairwise Wilcoxon Rank Sum tests. E. Spearman rank correlation between upregulation of CD70 expression upon CD40/TLR9 stimulation and baseline CD27+ B-cell frequency.

Figure 4. Impaired cytokine production by cirrhotic B-cells after CD40/TLR9 activation

A. TNFβ secretion upon CD40/TLR9 stimulation in HD, EF and CIR/HCC patients. Spearman rank correlations of B. TNFβ, C. TNFα, and D. IL-6 and CD27+ B-cell frequency. E. IgG, IgM, and IgA titers in CD40/TLR9 across patient groups compared by Kruskal-Wallis and pairwise Wilcoxon Rank Sum tests.

Figure 4. Impaired cytokine production by cirrhotic B-cells after CD40/TLR9 activation

A. TNFβ secretion upon CD40/TLR9 stimulation in HD, EF and CIR/HCC patients. Spearman rank correlations of B. TNFβ, C. TNFα, and D. IL-6 and CD27+ B-cell frequency. E. IgG, IgM, and IgA titers in CD40/TLR9 across patient groups compared by Kruskal-Wallis and pairwise Wilcoxon Rank Sum tests.

Figure 5. Reduced allostimulatory capacity of cirrhotic B-cells

A. Maximal CFSE dilution (calculated as gMFI of CFSE in T-cells co-cultured with activated B-cells minus gMFI of media-control T-cells divided by gMFI of anti-CD3/CD28-bead stimulated T-cells minus gMFI of media-control T- cells) across patient groups. Correlation of maximal CFSE dilution and B. baseline CD27+ B-cell frequency, C. frequency of CD70+ on post-CD40/TLR9 activation B-cells, D. post-activation B-cell HLA DR expression, E. post-activation B-cell CD86 expression, F. post-activation B-cell CD40 expression.

Figure 5. Reduced allostimulatory capacity of cirrhotic B-cells

A. Maximal CFSE dilution (calculated as gMFI of CFSE in T-cells co-cultured with activated B-cells minus gMFI of media-control T-cells divided by gMFI of anti-CD3/CD28-bead stimulated T-cells minus gMFI of media-control T- cells) across patient groups. Correlation of maximal CFSE dilution and B. baseline CD27+ B-cell frequency, C. frequency of CD70+ on post-CD40/TLR9 activation B-cells, D. post-activation B-cell HLA DR expression, E. post-activation B-cell CD86 expression, F. post-activation B-cell CD40 expression.

Figure 6. TLR4 and TLR9 activation of B-cells in cirrhosis

A. sCD14 plasma concentration by ELISA across patient groups. B. Inverse correlation of sCD14 concentrations and CD27+ B-cell frequency. C. Effect of cirrhotic versus healthy donor plasma on expression of HLA-DR, CD38, and CD19 on normal donor B-cells after 72 hours culture. Representative data from three separate experiments with different B-cell donors are shown. D. Impact of TLR4 (LPS-R. sphaeroides) and/or TLR9 (TTAGGG) antagonism of HD and CIR plasma on normal donor B-cells after 72 hours culture. Representative data from three separate experiments with different B-cell donors shown. E. Impact of three approaches to block TLR4 activation (LPS-R. sphaeroides, anti-CD14 and anti-TLR4) on the expression of HLA-DR on normal donor B-cells co-cultured with HD and CIR plasma. F.. Preservation of B-cell viability (lack of Live/Dead Aqua staining) by cirrhotic plasma and effect of TLR4 (LPS-R. sphaeroides) and/or TLR9 (TTAGGG) antagonism.

Figure 6. TLR4 and TLR9 activation of B-cells in cirrhosis

A. sCD14 plasma concentration by ELISA across patient groups. B. Inverse correlation of sCD14 concentrations and CD27+ B-cell frequency. C. Effect of cirrhotic versus healthy donor plasma on expression of HLA-DR, CD38, and CD19 on normal donor B-cells after 72 hours culture. Representative data from three separate experiments with different B-cell donors are shown. D. Impact of TLR4 (LPS-R. sphaeroides) and/or TLR9 (TTAGGG) antagonism of HD and CIR plasma on normal donor B-cells after 72 hours culture. Representative data from three separate experiments with different B-cell donors shown. E. Impact of three approaches to block TLR4 activation (LPS-R. sphaeroides, anti-CD14 and anti-TLR4) on the expression of HLA-DR on normal donor B-cells co-cultured with HD and CIR plasma. F.. Preservation of B-cell viability (lack of Live/Dead Aqua staining) by cirrhotic plasma and effect of TLR4 (LPS-R. sphaeroides) and/or TLR9 (TTAGGG) antagonism.

Figure 6. TLR4 and TLR9 activation of B-cells in cirrhosis

A. sCD14 plasma concentration by ELISA across patient groups. B. Inverse correlation of sCD14 concentrations and CD27+ B-cell frequency. C. Effect of cirrhotic versus healthy donor plasma on expression of HLA-DR, CD38, and CD19 on normal donor B-cells after 72 hours culture. Representative data from three separate experiments with different B-cell donors are shown. D. Impact of TLR4 (LPS-R. sphaeroides) and/or TLR9 (TTAGGG) antagonism of HD and CIR plasma on normal donor B-cells after 72 hours culture. Representative data from three separate experiments with different B-cell donors shown. E. Impact of three approaches to block TLR4 activation (LPS-R. sphaeroides, anti-CD14 and anti-TLR4) on the expression of HLA-DR on normal donor B-cells co-cultured with HD and CIR plasma. F.. Preservation of B-cell viability (lack of Live/Dead Aqua staining) by cirrhotic plasma and effect of TLR4 (LPS-R. sphaeroides) and/or TLR9 (TTAGGG) antagonism.