IFNγ/TNF responses to pAg are preserved in Vδ2+ γδT cells from CHB (but not AHB) subjects and are associated with their expression of Tbet/Eomes and NK markers but not PD1.

A. IFNγ/TNF responses to phosphoantigens in γδT-cells from CHB subjects. Representative FACS plots show IFNγ expression in γδT-cells in CD3-gated cells. Anti-Vγ9 TCR was used to detect Vδ2+ (Vγ9+) γδT-cells. Bar graphs show %IFNγ+, %IFNγ+/TNF+ and %TNF+ cells in CD3+ T-cells (white bars), Vδ1+ γδT-cells (blue bars) and Vδ2+ γδT-cells (red bars) from 23 CHB subjects, with PBMC stimulated for 1 day (23 hours) in-vitro with phosphoantigens zoledronic acid (zol) or (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) in addition to PMA/Ionomycin. Red bars on the far right provide comparisons to early IFNγ/TNF responses in Vδ2+ γδT-cells stimulated for 5 hours in separate assays. Cytokine responses between CD3+ T-cells, Vδ1+ γδT-cells and Vδ2+ γδT-cells were compared by non-parametric Kruskal Wallis test (k = 3). Comparisons between 2 conditions were made with non-parametric Mann Whitney U. *** p B. Comparisons between CHB, NC and AHB subjects for IFNγ/TNF responses in Vδ2+ γδT-cells to 1 day stimulation with phosphoantigens and PMA/Ionomycin stimulation. Bar graphs show %IFNγ+, %IFNγ+/TNF+ and %TNF+ cells in Vδ2+ γδT-cells following 1 day stimulation in-vitro with phosphoantigens zoledronic acid (zol) or (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) in addition to PMA/Ionomycin. Based on available cryopreserved PBMC, 23 CHB, 11 NC and 7 AHB subjects were included in this analysis. Error bars indicate 25% and 75% interquartile ranges. P-values between 3 groups were determined by Kruskal Wallis test (k = 3), followed by further two-way comparisons by Mann Whitney U for initial p-value below 0.05. P-values below 0.05 were considered significant and highlighted in red font for convenience. C. Correlations between IFNγ/TNF responses in Vδ2+ γδT-cells to zoledronic acid and their expression of T/NK markers. Scatter plots show %IFNγ+, %IFNγ+/TNF+ and %TNF+ cells in Vδ2+ γδT-cells (following 23 hours of stimulation) on the y-axis, with x-axis showing percent expression of various T/NK markers and Tbet/Eomes, combining results from 23 CHB, 11 NC and 7 AHB subjects. Correlation coefficient and p-values were determined by non-parametric Spearman rank order correlation test. For convenience, red font was used to indicate significantly positive correlations with p-values <0.05 whereas blue font was used to indicate significantly negative correlations with p-values <0.05.