Fig. 7: ZOL expands de novo Vγ9Vδ2 T_RM from blood, targeting ZOL-sensitised HCC cell lines.

a Representative flow cytometry plots of CD69⁺CD103⁺ and CD69⁺CD49a⁺ expression on Vγ9Vδ2 T-cells pre- and post-Zoledronic acid (ZOL) and IL-2 based expansion. b Summary data of CD69⁺CD103⁺ and CD69⁺CD49a⁺ expression on Vγ9Vδ2 T-cells pre- and post-ZOL and IL-2 based expansion (n = 15, p = 0.001). c % CXCR6 (left) and % CXCR3 (right) expression on ex vivo intrahepatic Vγ9Vδ2 T_RM (n = 11)_, compared to de novo blood CD69⁺CD103⁺ or CD69⁺CD49a⁺ Vγ9Vδ2 T_RM (induced T_RM, n = 8) following ZOL/IL-2 expansion (CXCR6 p = 0.0001, p = 0.0008; CXCR3 p = 0.0002, p = 0.0002). d IFN-γ expression (left) by ex vivo intrahepatic Vγ9Vδ2 T_RM compared to induced blood Vγ9Vδ2 T_RM after 4 h PMA and Ionomycin stimulation (n = 7, p = 0.001); unstimulated Granzyme B expression (right) by intrahepatic Vγ9Vδ2 T_RM (n = 11) compared to induced blood Vγ9Vδ2 T_RM (n = 6, p = 0.002, p = 0.001). e Schema demonstrating expanded blood Vγ9Vδ2 T-cells co-culture with untreated or ZOL pre-treated (5 μM, 16–18 h) HepG2 cells (E:T 2:1 ratio; 0.6 × 10⁶ expanded Vγ9Vδ2 T-cells to 0.3 × 10⁶ HepG2 cells, 6 h co-culture, all conditions performed in triplicate). Representative flow cytometry plot and summary data of IFN-γ and TNF-α expression by ZOL/IL-2 expanded Vγ9Vδ2 T-cells: unstimulated, directly treated with ZOL 5 μM, or after co-culture with untreated or ZOL pre-treated HepG2 cells (n = 10; IFN-γ p < 0.0001, p = 0.03, p = 0.01; TNF-α p < 0.001, p = 0.03, p = 0.01). f IFN-γ and TNF-α expression by ZOL/IL-2 expanded Vγ9Vδ2 T-cells after co-culture with ZOL pre-treated HepG2 cells, with or without the addition of 100 µM Mevastatin (Mev) (n = 10; IFN-γ p = 0.004, TNF-α p = 0.004). g IFN-γ and Granzyme B expression by expanded blood Vγ9Vδ2 T_RM (n = 9) compared to ex vivo intrahepatic Vγ9Vδ2 T_RM (n = 10) after co-culture with ZOL pre-treated HepG2 cells (IFN-γ p = 0.03, GRZB p = 0.005). h IFN-γ and TNF-α expression by ZOL/IL-2 expanded Vγ9Vδ2 T-cells after co-culture with ZOL pre-treated HepG2 cells, with or without the addition of anti-programmed death-ligand 1 (PDL-1) blockade (n = 6). i Lysis of HepG2 cells and ZOL pre-treated HepG2 cells after co-culture with PBMCs containing ZOL-expanded Vγ9Vδ2 T-cells (n = 7, p = 0.02), measured using Toxilight^TM cytotoxicity assay. Each symbol represents a study participant; error bars show mean ± SEM. Two-tailed p-values determined using Wilcoxon paired test (b, f, i), Mann–Whitney test (c, d, g), or Friedman test with Dunn’s post-hoc test for multiple comparisons (e, h). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.