Fig 2: HBsAg dysregulate IL-21 secretion by TFH cells but do not obstruct IL-27 production. (A) Representative flow cytometry plot and collective data in bar graphs illustrate HBV-specific IL-21 expressing TFH cells after stimulation with PepMix HBV large envelop protein Ultra (HBs) which has a mix of 216 peptides (15mers with 11 aa overlap) and HBV capsid/core protein which has a pool of 44 peptides, at a concentration of 1 μg/ml for 5 days in the presence of CD49d and CD28 (2 μg/ml), re-stimulation with HBs and HBcAg was done on day 4. Global IL-21 expressing cells were measured after overnight PMA/ionomycin stimulation followed by intracellular cytokine staining and subsequent data analysis by flowJo. Cells without any stimulation were taken as controls. (B). IL-21 was detected by multiplex cytokine bead array assay in cell supernatant collected after TFH cell sorting and following stimulation with PMA/ionomycin as well as in plasma. (C) Analysis of IL-21 Relative mRNA expression in sorted TFH cells (D) HBV-specific and global IL-27p28/EBI3, IL-27p28, and IL-27EBI3 producing cells were assessed by CD4 T cells after 5 days stimulation with HBs, HBc peptides and PMA/ionomycin as mentioned above. (E) IL-27p28/EBI3 heterodimer production was also evaluated specifically in sorted TFH cells supernatant stimulated with PMA/ionomycin and relative mRNA expression was analyzed in sorted TFH cells (F) IL-27p28/EBI3 heterodimer was also measured in the plasma (G) HBV-specific as well as global IL-27p28/EBI3, IL-27p28, and IL-27EBI3 producing TFH cells directed against HBs and HBc peptides and PMA/ionomycin. (H) Comparison of IL-21 vs. IL-27p28/EBI3 heterodimer expressing TFH cells in CHB patients; HBsAg-specific cytokine producing cells has been shown in representative concatenate flow cytometry plot and cumulative data has been demonstrated in bar graphs where IL-21 expressing TFH cells were compared with IL-27p28/EBI3 heterodimer as well as IL-27p28 and IL-27EBI3. All the flow cytometry cytokine analysis was performed in fourteen CHB and HC-vacc in each group. To measure cytokine in sorted TFH cells supernatant, four HC-vacc and three CHB patients were taken. Plasma cytokines were detected in nine CHB and HC-vacc in each group. Bars indicate median with range or mean with standard deviation. P values were determined either by non-parametric, 2-tailed Mann-Whitney U test or unpaired t test.

Fig 3: 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 4: 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.

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.