Fig. 6: Anti-tumour potential of Vγ9Vδ2 T_RM against ZOL-sensitised HCC cell lines.

a Schema of untreated or Zoledronic acid (ZOL) pre-treated (5μM, 16–18 h) adherent HepG2 cells co-cultured with intrahepatic lymphocytes (IHL) (n = 11) or HCC tumour-infiltrating lymphocytes (TIL) (n = 4) (E:T 2:1 ratio; 0.6 × 10⁶ IHL or TILs to 0.3 × 10⁶ HepG2 cells, 6 h co-culture, all conditions performed in duplicate or triplicate). b IFN-γ, TNFα expression by Vγ9Vδ2 T-cells in IHL unstimulated or after co-culture with untreated HepG2 cells (n = 11 IFN-γ, n = 8 TNF-α). c IFN-γ, TNFα, IL-2 expression by Vγ9Vδ2 T-cells in IHL: unstimulated, directly treated with 5 μM ZOL, or after co-culture with untreated or ZOL pre-treated HepG2 cells (n = 11 IFN-γ p = 0.0004, p = 0.0008; n = 8 TNFα p = 0.0006, p = 0.004; n = 7 IL-2 p = 0.007, p = 0.02). d IFN-γ (n = 11), TNF-α (n = 8) expression by Vγ9Vδ2 T-cells in IHL after co-culture with ZOL pre-treated HepG2 cells with or without 100 µM Mevastatin (Mev) treatment (IFN-γ p = 0.008, TNF-α p = 0.03). e Representative flow cytometry plot of IFNγ expression and summary data of IFNγ and IL-2 expression by CD69⁺CD49a⁺ Vγ9Vδ2 T_RM compared to CD69⁻CD49a⁻ Vγ9Vδ2 non-T_RM, following co-culture of IHL with ZOL pre-treated HepG2 cells (g = 8, IFNγ p = 0.02, IL-2 p = 0.03). f IFN-γ expression by Vγ9Vδ2 T-cells in HCC TILs unstimulated, or after co-culture with untreated or ZOL pre-treated HepG2 cells (n = 4). g Representative flow cytometry plot of IFN-γ expression by CD69⁺CD49a⁺ Vγ9Vδ2 T_RM and CD69⁻CD49a⁻ Vγ9Vδ2 non-T_RM in HCC TILs after co-culture with ZOL pre-treated HepG2 cells. Each symbol represents a study participant; error bars represent the mean ± SEM. Two-tailed p-values were determined using Wilcoxon paired test (b, d, e), or Friedman test with Dunn’s post-hoc multiple comparisons test (c, f). *p < 0.05; **p < 0.01; ***p < 0.001.