Circulating and intrahepatic antiviral B cells are defective in hepatitis B
Alice R. Burton, Laura J. Pallett, Laura E. McCoy, Kornelija Suveizdyte, Oliver E. Amin, Leo Swadling, Elena Alberts, Brian R. Davidson, Patrick T.F. Kennedy, Upkar S. Gill, Claudia Mauri, Paul A. Blair, Nadege Pelletier, Mala K. Maini
Alice R. Burton, Laura J. Pallett, Laura E. McCoy, Kornelija Suveizdyte, Oliver E. Amin, Leo Swadling, Elena Alberts, Brian R. Davidson, Patrick T.F. Kennedy, Upkar S. Gill, Claudia Mauri, Paul A. Blair, Nadege Pelletier, Mala K. Maini
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Research Article Immunology Infectious disease

Circulating and intrahepatic antiviral B cells are defective in hepatitis B

Abstract

B cells are increasingly recognized as playing an important role in the ongoing control of hepatitis B virus (HBV). The development of antibodies against the viral surface antigen (HBV surface antigen [HBsAgs]) constitutes the hallmark of resolution of acute infection and is a therapeutic goal for functional cure of chronic HBV (CHB). We characterized B cells directly ex vivo from the blood and liver of patients with CHB to investigate constraints on their antiviral potential. Unexpectedly, we found that HBsAg-specific B cells persisted in the blood and liver of many patients with CHB and were enriched for T-bet, a signature of antiviral potential in B cells. However, purified, differentiated HBsAg-specific B cells from patients with CHB had defective antibody production, consistent with undetectable anti-HBs antibodies in vivo. HBsAg-specific and global B cells had an accumulation of CD21–CD27– atypical memory B cells (atMBC) with high expression of inhibitory receptors, including PD-1. These atMBC demonstrated altered signaling, homing, differentiation into antibody-producing cells, survival, and antiviral/proinflammatory cytokine production that could be partially rescued by PD-1 blockade. Analysis of B cells within healthy and HBV-infected livers implicated the combination of this tolerogenic niche and HBV infection in driving PD-1hiatMBC and impairing B cell immunity.

Authors

Alice R. Burton, Laura J. Pallett, Laura E. McCoy, Kornelija Suveizdyte, Oliver E. Amin, Leo Swadling, Elena Alberts, Brian R. Davidson, Patrick T.F. Kennedy, Upkar S. Gill, Claudia Mauri, Paul A. Blair, Nadege Pelletier, Mala K. Maini

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Figure 5

atMBC accumulating in CHB have impaired signaling and antiviral function.

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atMBC accumulating in CHB have impaired signaling and antiviral function...
(A) Representative flow cytometric analysis of Ca2+ influx (Fluo-4 AM; median fluorescence intensity) over time (seconds) in purified B cells after stimulation with F(ab′)2-IgG/IgA/IgM (anti-BCR; 50 μg/ml) or ionomycin (iono.) (1 μg/ml) (n = 10 patients with CHB). Basal fluorescence prior to stimulation is shaded gray. Summary plot: difference in MFI upon stimulation in cMBC and atMBC compared with baseline. (B) Expression of phosphorylated-BLNK (MFI) in global B cells after crosslinking with F(ab′)2-IgG/IgA/IgM for 30 seconds (anti-BCR; n = 8). Background fluorescence from paired stimulated control is shown in gray. (C–D) Intracellular cytokine staining for (C) IL-6 and (D) TNF-α in atMBC and cMBC after stimulation with F(ab′)2-IgG/IgA/IgM and CD40L (anti-BCR; soluble CD40L [sCD40L]; n = 10 patients with CHB) or R848 (resiquimod; TLR7/8 agonist; 1 μg/ml; n = 35 patients with CHB) for 24 hours. Frequencies are presented minus paired unstimulated control. (E) Anti-HBs–secreting B cells in unexposed controls (n = 5), vaccinated HC (n = 7), and patients with CHB (n = 14), determined by ELISpot. SFC, spot-forming cells. (F) atMBC were FACS sorted (n = 7 HBV-vaccinated HC) and differentiated into plasma cells alongside a matched number of cMBC. Graph shows proportion of cells acquiring a plasma cell phenotype (CD45+CD19+CD3–IgD–CD38hiCD20–CD27+CD138+), as determined by flow cytometry. (G) Ex vivo staining for annexin V on purified B cells, stratified by subset and by PD-1 expression, after stimulation with F(ab′)2-IgG and -IgM (1 μg/ml) and CD40L (0.5 μg/ml) for 7 days (n = 7). (H) Annexin V expression on B cells stimulated ± anti–PD-1 mAb (10 μg/ml) for 7 days (n = 7). (I) Intracellular staining for IL-6 on atMBC and cMBC stimulated as in G and H for 24 hours (n = 18). Frequency is presented minus paired unstimulated control. Error bars indicate mean ± SEM. P values were determined by Wilcoxon’s paired t test (A–D, F–I); and Kruskal-Wallis test (ANOVA) with Dunn’s post hoc test for pairwise multiple comparisons (E). *P < 0.05; **P < 0.005; ***P < 0.001; ****P < 0.0001.