(A) mRNA expression of PD-1 in the indicated B cell subsets of 5 CHB patients measured by NanoString. (B) Surface PD-1 expression on the B cell subsets of 96 CHB patients measured by flow cytometry. (C) Flow cytometric data of MBCs from 141 samples analyzed by UMAP and concatenated. Four subsets of MBCs were delineated (left, plots reshown from Figure 6A), and PD-1⁺ MBCs are shown (right). (D) MBCs of 15 healthy vaccinated (left) and 76 CHB patients (right) were downsampled to equal cell numbers. Density UMAP plots are shown; the cluster of AtM B cells is highlighted in red. Right bar graph shows percentage of global MBCs with an AtM phenotype in 17 healthy vaccinated and 96 CHB patients. (E) Double-positive HBsAg-D550⁺D650⁺ B cells from 15 healthy vaccinated (left) and 76 CHB patients (right) were concatenated, downsampled to normalized frequencies, and overlaid onto the UMAP plot of global concatenated MBCs; the cluster of AtM B cells is highlighted in red. Right bar graph shows percentage of HBsAg-specific B cells with an AtM phenotype in healthy vaccinated and CHB patients. (F) MFI of PD-1 on global MBCs and HBsAg-specific MBCs of 96 CHB patients. (G) Double-positive HBsAg-D550⁺D650⁺ MBCs from 4 CHB patients and 3 healthy vaccinated subjects were FACS sorted and cocultured for 13 days with CD40L-expressing feeder cells in the presence of IL-2 and IL-21, with or without anti–PD-1 Ab (schematic at left). Subsequently, anti-HBs–secreting cells were detected by ELISpot assay (right). Average fold changes in the number of anti-HBs spots are shown above the plots. Data are presented as median, and statistical analysis was performed by the Mann-Whitney U test (D and E) and Wilcoxon’s paired t test (F and G). *P < 0.05; **P < 0.01; ****P < 0.0001.