BACKGROUND & AIMS Knowledge about the regulation ofanti-HBV humoral immunity during natural HBV infection is limited. We recently utilized dual fluorochrome-conjugated HBsAg to demonstrate, in patients with chronic HBV (CHB) infection, the functional impairment of their HBsAg-specific B cells. However, the features of their HBcAg-specific B cells are unknown. Here we developed a method to directly visualize, select and characterize HBcAg-specific B cells in parallel with HBsAg-specific B cells. METHODS Fluorochrome-conjugated HBcAg reagents were synthetized and utilized to detect directly ex vivo HBcAg-specific B cells in 36 CHB patients. The frequency, phenotype, functional maturation and transcriptomic profile of HBcAg-specific B cells was studied by flow cytometry, in vitro maturation assays and Nanostring based detection of expression of immune genes, which we compared with HBsAg-specific B cells and total B cells. RESULTS HBcAg-specific B cells are present at higher frequency than HBsAg-specific in CHB patients and, differently to HBsAg-specific B cells, they mature efficiently into antibody-secreting cells in vitro. Their phenotypic and transcriptome profiles show that HBcAg-specific B cells are preferentially IgG+ memory B cells.However, despite their phenotypic and functional differences, HBcAg- and HBsAg-specific B cells of CHB patients share a mRNA expression pattern thatdiffers from global memory B cells and is characterized by high expression of genes indicative of cross-presentation and innate immune activity. CONCLUSIONS During chronic HBV infection, a direct relation exists between serological detection of anti-HBs and anti-HBc antibodiesand quantity and function of their respective specific B cells. However, the transcriptomic analysis performed in HBsAg- and HBcAg-specific B cells suggests additional roles of HBV-specific B cells beyond the production of antibodies. LAY SUMMARY Protection of viral infection necessitates the production of antibodies that are generated by specialized cells of the immune system called B cells. During chronic HBV infection, antibodies against the internal part of the virus (core or HBcAg) are detectable while the antibodies directed against the virus envelope (surface or HBsAg) are not present. Here we developed a method that allows us to directly visualize ex vivo the B cells specific for these two viral components, highlighting their differences and similarities, and showing how two components of the same virus can differently impact on the function of antiviral B cells.

中文翻译：

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慢性乙型肝炎患者HBV核衣壳和包膜蛋白特异性B细胞的比较表征

背景与目的 关于自然 HBV 感染期间抗 HBV 体液免疫调节的知识是有限的。我们最近使用双荧光染料偶联的 HBsAg 来证明慢性 HBV (CHB) 感染患者的 HBsAg 特异性 B 细胞功能受损。然而，其 HBcAg 特异性 B 细胞的特征尚不清楚。在这里，我们开发了一种与 HBsAg 特异性 B 细胞并行直接可视化、选择和表征 HBcAg 特异性 B 细胞的方法。方法 合成荧光染料偶联的 HBcAg 试剂并用于直接检测 36 名 CHB 患者的离体 HBcAg 特异性 B 细胞。通过流式细胞术研究了 HBcAg 特异性 B 细胞的频率、表型、功能成熟和转录组学特征，体外成熟试验和基于纳米线的免疫基因表达检测，我们将其与 HBsAg 特异性 B 细胞和总 B 细胞进行了比较。结果 HBcAg 特异性 B 细胞在 CHB 患者中的出现频率高于 HBsAg 特异性，并且与 HBsAg 特异性 B 细胞不同，它们在体外有效成熟为抗体分泌细胞。他们的表型和转录组图谱表明，HBcAg 特异性 B 细胞优先为 IgG+ 记忆 B 细胞。然而，尽管它们的表型和功能不同，但 CHB 患者的 HBcAg 和 HBsAg 特异性 B 细胞具有与全局记忆 B 细胞不同的 mRNA 表达模式并且特征在于指示交叉呈递和先天免疫活性的基因的高表达。结论 在慢性 HBV 感染期间，抗-HBs和抗-HBc抗体的血清学检测与其各自特异性B细胞的数量和功能存在直接关系。然而，在 HBsAg 和 HBcAg 特异性 B 细胞中进行的转录组学分析表明，除了产生抗体之外，HBV 特异性 B 细胞还有其他作用。概述 病毒感染的保护需要产生抗体，这些抗体是由免疫系统的特殊细胞（称为 B 细胞）产生的。在慢性 HBV 感染期间，可检测到针对病毒内部部分（核心或 HBcAg）的抗体，而针对病毒包膜（表面或 HBsAg）的抗体不存在。在这里，我们开发了一种方法，允许我们直接在体外可视化这两种病毒成分的特异性 B 细胞，