Expansion of Human Peripheral Blood γδ T Cells using Zoledronate

Summary

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.

Abstract

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

Protocol

1. Isolation of PBMC

1. Draw blood (7.5-8.0 ml) into a BD Vacutainer CPT Cell Preparation Tube with Sodium Heparin. The tube contains a sodium heparin anticoagulant and a Ficoll-Hypaque density fluid, plus a polyester gel barrier, which separates the two liquids. Centrifuge tube/blood sample at room temperature (18°C to 25°C) in a horizontal rotor (swing-out head) for 20 min at 1800 x g. Switch centrifuge brakes off.
2. After centrifugation, the sequence of layers occurs as follows (seen from top to bottom): a) plasma - b) peripheral blood mononuclear cells (PBMC) and platelets - c) density solution - d) polyester gel - e) granulocyt....

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
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