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. 2020 Feb 4;10:1835. doi: 10.1038/s41598-020-58870-2

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Analysis of HBsAg-specific B cell responses by HBsAg-specific ELISpot assay. PBMCs were stimulated with R848 and rIL-2 for 5 days and HBsAg-specific IgG as well as total IgG antibody-secreting cells (ASCs) were assessed by HBsAg-specific ELISpot assay. (A) Representative ELISpot image for HBsAg-specific or total IgG ASCs from the HBs^lo (designated as lo) or HBs^hi (designated as hi) patients. Number of (B) HBsAg-specific IgG and (C) total IgG ASCs per 10⁶ PBMCs from the HBs^lo (n = 21) and HBs^hi (n = 13) groups. (D) Fold change of HBsAg-specific IgG ASCs by R848 + αPD-1 antibody with respect to R848 stimulation alone, between the HBs^lo and HBs^hi groups. (E) Fold change of HBsAg-specific IgG ASCs by CpG-B + αPD-1 with respect to CpG-B stimulation alone, between patients with HBsAg < 100 IU/ml and HBsAg > 5,000 IU/ml by ELISpot assay. Pi-charts represent the proportion of patients ≥ 2-fold change (Responder, Dark) and those <-2-fold change (non-responder) between 2 groups. Each data point represents 1 sample and horizontal line represents the median value. Mann-Whitney U test were performed for statistical analysis. *p <0.05. χ²-test were performed to assess an association between serum HBsAg level and fold changes to αPD-1 antibody. τ; p < 0.05 in χ²-test.