A method to expand γδ T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived γδ T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of γδ T cells can be applied to autologous cellular immunotherapy of cancer.
Human γδ T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of γδ T cells in peripheral blood have the Vγ9Vδ2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, γδ T cells are attractive candidate effector cells for cancer immunotherapy. Vγ9Vδ2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of γδ T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and γδ T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to γδ T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating γδ T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate Vγ9Vδ2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of γδ T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of γδ T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of γδ T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded γδ T cells display CD27–CD45RA– effector memory phenotype and thier function can be evaluated by IFN-γ production assay. 7
The method presented here enables efficient expansion of γδ T cells from PBMC. γδ T cells activated and expanded by zoledronate and IL-2 develop complete effector functions, reflected by cytokine production and cytotoxicity. It has been reported that the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP) and 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP) also expand γδ T cells; however, they are not commercially available. In contrast, zoledronate is already licensed for clinica…
The authors have nothing to disclose.
Reagent name | Company | Catalogue number | Comments (optional) |
---|---|---|---|
ZOMETA | Novartis Pharma K. K | zoledronate | |
PROLEUKIN | Novartis Pharmaceuticals | human recombinant IL-2 | |
BD Vacutainer CPT Cell Preparation Tube with Sodium Heparin | BD | 362753 | |
RPMI1640 | Invitrogen | 21870-076 | |
ALyS203- medium | Cell Science & Technology Institute | 0301-7 | |
OpTmizer | Invitrogen | 0080022SA | |
brefeldin A | Sigma | B5936-200UL | |
phorbol 12-myristate 13-acetate (PMA) | Sigma | P1585-1MG | |
ionomycin | Sigma | 13909-1ML | |
IntraPrep | BECKMAN COULTER | A07803 | |
anti-human CD3-FITC or PE/Cy5 | BECKMAN COULTER | A07746 FITC A07749 PE/Cy5 |
|
anti-human CD4-ECD | BECKMAN COULTER | 6604727 | |
anti-human CD8-PE/Cy5 | BECKMAN COULTER | 6607011 | |
anti-human CD14-PE/Cy5 | BECKMAN COULTER | A07765 | |
anti-human CD19-PE | BECKMAN COULTER | A07769 | |
anti-human CD45-ECD | BECKMAN COULTER | A07784 | |
anti-human CD56-PE/Cy5 | BECKMAN COULTER | A07789 | |
anti-human TCRαβ-PE | BECKMAN COULTER | A39499 | |
anti-human TCR Vγ9-FITC | BECKMAN COULTER | IM1463 | |
anti-human CD27-PE/Cy5 | BECKMAN COULTER | 6607107 | |
anti-human CD45RA-ECD | BECKMAN COULTER | IM2711 | |
anti-human CD69-PE | BD | 555531 | |
anti-human NKG2D-PE | BECKMAN COULTER | A08934 | |
Anti-humal IFNγ-PE | BECKMAN COULTER | IM2717U | |
Mouse IgG1 isotype control-PE | BECKMAN COULTER | A07796 | |
Mouse IgG1 isotype control-ECD or PE/Cy5 | BECKMAN COULTER | A07797 A07798 |