IL-12 conditioning of peripheral blood mononuclear cells from breast cancer patients promotes the zoledronate-induced expansion of γδ T cells in vitro and enhances their cytotoxic activity and cytokine production

Abstract

Background: In a series of our preclinical studies, we have reported that conditioning of α/β CD8⁺ T cells in vitro with interleukin-12 (IL-12) during their expansion improves their homing phenotype and anti-tumor cytolytic function upon their adoptive transfer in vivo. Vγ9⁺Vδ2⁺ T cells can also be expanded in vitro with amino bisphosphonates such as zoledronate (ZOL) for the purpose of adoptive therapy.

Aim: We aimed in this study to use IL-12 to enhance the expansion and cytotoxic functions of ZOL-expanded Vγ9⁺Vδ2⁺T cells.

Materials and methods: Peripheral blood mononuclear cells (PBMCs) were separated from healthy donors and stage II breast cancer patients. PBMCs (1 × 10⁶ cells/mL) were cultured and treated with ZOL/IL2, ZOL/IL2/IL12, or IL2/IL12. Cultured cells were harvested on days 7 and 14 of culture and their numbers, phenotype, and cytolytic activity were assessed. The levels of pro- and inflammatory cytokines/chemokines in the plasma and supernatants of the cultured cells were analyzed by Luminex.

Results: In healthy subjects, the addition of IL-12 to ZOL/IL2-stimulated PBMCs increased the expansion and the cytotoxic activity of Vγ9⁺Vδ2⁺ T cells on days 7 and 14 of culture. The latter was measured by the expression level of the cytolytic molecules granzyme B (GZB) and perforin (PER). Of note, αβ CD8 + T cells were also activated under the same condition but with a lesser extent addition of IL-12 to ZOL/IL2-stimulated PBMCs from cancer patients also induced similar effects but were lower than in control subjects. Interestingly, ZOL/IL2/IL12-treated PBMCs showed higher levels of cytokines/chemokines, in particular, CCL, CCL4, GM-CSF, IL-1rα; IL-12, IL-13, TNF, and IFNγ measured on days 7 and 14.

Conclusion: The addition of IL12 at the start of the expansion protocol can enhance the activity of γδ T cells which might be mediated in part by the activation of αβ T cells.

Keywords: Chemokines; Cytokines; Granzyme B; IL-12; IL-2; Perforin; Zoledronate; αβ T cells; γδ T cells.