Fig 2: Distinct and common transcriptional response of IE-CTLs in response to tissue alarmins and adaptive cytokine. After 12 days in culture, IE-CTLs were starved for 48 h, then stimulated for 30 min, 3 h, 4 h or 24 h with IFNβ, IL-15 or IL-21. Unstimulated cells (0 h) were used as control. n = 8 samples per time point and condition. The transcriptome was analyzed by RNA-seq. (A) Number of DEGs after cytokine treatment in comparison with the unstimulated samples at each time point (|log2 fold change|>1, FDR ≤ 0.01). Light colors indicate up-regulated genes. Dark colors indicate down-regulated genes. (B) Centroids of all the samples using Principal Components 1 and 2 from all DEGs reveal different time course patterns of gene expression upon IFNβ (green), IL-15 (orange) or IL-21 (purple) stimulation on IE-CTLs. (C) Venn diagram depicting the number of unique and overlapping genes across the stimulations and time points. The most significantly enriched pathways (determined by Reactome) of shared genes between the three stimulations are depicted. Color (key) indicates level of significance. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 3: S703I JAK1 Mediates Hyperactive STAT Phosphorylation by Transactivation of Partnering JAKs(A) Immunoprecipitation of JAK1 from JAK1-transduced U4C cells and immunoblotting for total phosphorylation (4G10) after stimulation with IFN-α (100 IU/mL) for 15 min.(B) Immunoprecipitation of JAK2 from patient-derived B-EBV cells and western blotting for phosphorylation at the activation loop after stimulation with IFN-γ (0.1 ng/mL) for 15 min.(C) Immunoprecipitation of TYK2 from JAK1-transduced U4C cells and western blotting for phosphorylation at the activation loop after stimulation with IFN-α (100 IU/mL) for 15 min.(D) Immunoprecipitation of JAK3 from patient-derived B-EBV cells and western blotting for total phosphorylation stimulation with IL-21 (50 ng/mL) for 15 min.(E) Surface staining of the IFNAR2 subunit by flow cytometry in U4C cells transduced with the indicated constructs. Lines indicate means and error bars represent SD. ∗∗∗p < 0.001, ****p < 0.0001, analysis of variance (ANOVA) with Tukey's post-hoc analysis.(F and G) Transduction of U4C cells with catalytically inactivated JAK1 (K908A), WT JAK1, S703I JAK1, or double-mutant JAK1 (K908A S703I), followed by stimulation with IFN-α (1,000 IU/mL) (F) or IFN-γ (1.0 ng/mL) (G).(H) Transduction of U4C cells with catalytically inactivated JAK1 (K908A), WT JAK1, A634D JAK1, or double-mutant JAK1 (K908A A634D), followed by stimulation with IFN-α (1000 IU/mL) or IFN-γ (1.0 ng/mL).(I) Inhibition of STAT phosphorylation by a 4-h treatment with saturating doses (1 and 10 μM) of JAK inhibitors followed by 15-min stimulation with IFN-α (1,000 IU/mL).All of the data are representative of at least 3 independent experiments.See also Figure S2.

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: Mechanism of TFH mediated B cells help. HBV infection impair IL-21 production by TFH cells without obstructing IL-27 secretion. IL-27 binds to its cognate receptor present on the surface of B cells, activate downstream signaling, and further induces Blimp-1 expression and lead to the formation of plasmablasts and plasma cells resulting in HBsAg-specific and total antibody secretion.