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: SARS-CoV-2-specific CD4+ T cell responses of healthy, severely ill, and recovered individuals(A) Representative flow cytometry plots showing the peptide-induced expression of CD40L, IFN?, IL-4/13, and IL-21 in memory CD4+ T cells for each group of individuals.(B) Compared are the frequencies of CD4+ T cells specific for S, N, and M proteins between healthy, severely ill, and recovered individuals. Each of the graphs represents one of the four CD4+ T cell helper functions investigated (expression of CD40L, IFN?, IL-4/13, and IL-21).(C) Comparison of CD4+ T cell frequencies responding to S-, N-, and M-based peptide pools.(D) SPICE analysis of SARS-CoV-2-specific CD4+ T cell responses. Each pie chart depicts the prevalence of polyfunctional subsets within a certain group of individuals for a particular protein stimulus. Arcs surrounding the pie charts show the relative contribution of each of the four investigated CD4+ T cell functions. The number of independent experiments represented in the above graphs is n = 8 for healthy, n = 13 for severely ill, and n = 18 for the recovered group. Data are presented as mean ± SD. Differences between the groups were assessed using the Mann-Whitney test or Wilcoxon test for matched data. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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.