Fig 2: To trigger CD8+ T and NK cell proliferation, costimulation by 4‐1BBL is superior to CD28. Proliferation of MART‐1‐specific HLA‐A*02‐restricted CD8+ T cells in the presence of peptide P26‐35 (500 ng/ml), cytokines (IL‐15 + IL‐21), and the indicated concentrations of: (A) soluble anti‐4‐1BB antibody clone 4B4‐1 (circles) or mIgG1 isotype controls (squares) (n = 5); (B) soluble anti‐4‐1BB antibody clone BBK‐2 (circles) or mIgG1 isotype controls (squares) (n = 5); (C) soluble anti‐4‐1BB antibody clone Urelumab (circles) or hIgG4 isotype controls (squares) (n = 5); and (D) soluble anti‐CD28 antibody (circles) or mIgG1 isotype control (squares) (n = 5). Proliferation of freshly purified NK cells in the presence of cytokines (IL‐15 + IL‐21), the absence (green symbols) or presence of either CD86/4‐1BBL‐expressing (red symbols) or parental (blue symbols) K562, and the indicated concentrations of: (E) soluble anti‐4‐1BB antibody clone 4B4‐1 (circles) or mIgG1 isotype controls (squares) (n = 28); (F) soluble anti‐4‐1BB antibody clone BBK‐2 (circles) or mIgG1 isotype controls (squares) (n = 11); (G) soluble anti‐4‐1BB antibody clone Urelumab (circles) or hIgG4 isotype controls (squares) (n = 15); and (H) soluble anti‐CD28 antibody (circles) or mIgG1 isotype controls (squares) (n = 10). Data is shown as the mean ± SEM. Normalization of the data and statistical analysis were performed as described in the Section Methods, 2.

Fig 3: Specific epigenomic profiles induced by alarmins and adaptive cytokine. All the differential H3K27ac peaks after 3 h of stimulation with IFNβ, IL-15 or IL-21 (FDR ≤ 0.01) are depicted in (A) barplots and (B) used for PCA to depict epigenetic responses of IE-CTLs upon stimulation. (C) Scatter plot displaying the association between changes in H3K27ac (Y-axis) and expression (X-axis) upon stimulation. Gene-peak pairs were grouped based on the direction of the fold change in expression (Exp+ and Exp-) and the direction (or lack of changes) in H3K27ac after stimulation (Epi+, Epi - and Epi NA). Red dots (Epi + Exp+) indicate a fold change >1 in both expression and H3K27ac occupancy. Green dots (Epi- Exp-) indicate a fold change < 1 in both expression and H3K27ac occupancy. Dark blue (Exp + EpiNA) and light blue (Exp- EpiNA) are gene-peak pairs with fold change >1 or <1 in expression, respectively, and no change in H3K27ac. (D) Violin plots showing the distribution of gene expression levels (left, log2(VST+1)) and H3K27ac occupation (right, log2(Counts+1)) in up-regulated genes responding to IFNβ (upper panel) and IL-15 (lower panel) stimulation. Upregulated genes with H3K27ac |fold change| >1 (Epi+/Exp+) are shown in red and those without H3K27ac changes (EpiNA/Exp+) are shown in blue. Significant differences were assessed by Wilcoxon test (*p < 0.05; ***p < 0.01; ****p < 0.001, NS non-significant). (E) Representative H3K27ac tracks illustrating the concordance between epigenomics and gene expression after IFNβ or IL-15 treatment. n = 8 samples per time point and condition. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 4: IL-27 secreted by TFH cells support plasmablasts and plasma cell formation. (A–C) Mechanism of TFH mediated B cell response was determined by autologous TFH-B cell co-culture (CHB: n=5, HC-vacc: n=5). To imitate HBV-specific interactions between TFH and B cells, FACS-sorted TFH cells were first primed with HBsAg for 3 h, washed and then incubated with autologous CD19+CD27+ memory and CD19+CD27-IgD+ naïve B cells with and without of IL-21 and IL-27 neutralizing antibodies for 5 days. Generation of plasmablasts and plasma cells was analyzed by flow cytometry (D) expression of Blimp-1 (CHB: n=5, HC-vacc: n=5). Plasmablasts were gated as CD27+CD38+ cells and plasma cells were defined based on CD27+CD38+CD138. (E) Incubation of memory B and naïve B cells with rIL-27 for 5 days showed increased plasmablasts and plasma cell formation (CHB: n=5). Statistical analysis was performed using either Kruskal-Wallis test (ANOVA) with Dunn’s post hoc test for multiple comparisons or paired t test. Bars indicates mean and error bars designate standard deviation. * indicates p < 0.05, **p < 0.01 and ***p < 0.001.

Fig 5: IL-27 help B cells for HBsAg-specific and total antibody secretion. (A) FACS-purified TFH cells were first primed with HBsAg for 3 h, washed and then cultured with autologous memory and naïve B cells in the presence and absence IL-21 and IL-27 neutralizing antibody for 5 days. Supernatant was harvested and IgG, IgM and IgA were quantified by ELISA (CHB: n=5, HC-vacc: n=5). (B-E) Representative ELISpot image indicate HBsAg-specific as well as total IgG, IgM and IgA secreting cells in HC-vacc and CHB patients after 5 days culture in the presence of rIL-21, rIL-27, R848+IL-2 alone and in different combinations. No stimulation was given in controls. Bar graphs shows cumulative data of six subjects in each group and indicate mean of the no. of antigen specific as well as total antibody producing cell spot. P values were determined by Kruskal-Wallis test (ANOVA) with Dunn’s post hoc test for multiple comparisons or paired t test. Bars represent mean with standard deviation. Data comparisons were made between controls (without any stimulation) of HC-vacc and CHB with different stimulations and p values are flagged with *. Comparisons between HC-vacc and CHB were also analyzed and p values are flagged with ♦. *,♦ indicates p<0.05, **,♦♦ p<0.01 and ***,♦♦♦ p<0.001. (F) Levels of plasma antibodies were determined by ELISA and significance was calculated by non-parametric, 2-tailed Mann-Whitney U test.