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) granulocytes - f) re....

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

List of materials used in this article

Name	Company	Catalog Number	Comments
ZOMETA	Novartis AG		zoledronate
PROLEUKIN	Novartis AG		human recombinant IL-2
BD Vacutainer CPT Cell Preparation Tube with Sodium Heparin	BD Biosciences	362753
RPMI1640	Invitrogen	21870-076
ALyS203- medium	Cell Science & Technology Institute	0301-7
OpTmizer	Invitrogen	0080022SA
brefeldin A	Sigma-Aldrich	B5936-200UL
phorbol 12-myristate 13-acetate (PMA)	Sigma-Aldrich	P1585-1MG
ionomycin	Sigma-Aldrich	13909-1ML
IntraPrep	Beckman Coulter Inc.	A07803
anti-human CD3-FITC or PE/Cy5	Beckman Coulter Inc.	A07746 FITC A07749 PE/Cy5
anti-human CD4-ECD	Beckman Coulter Inc.	6604727
anti-human CD8-PE/Cy5	Beckman Coulter Inc.	6607011
anti-human CD14-PE/Cy5	Beckman Coulter Inc.	A07765
anti-human CD19-PE	Beckman Coulter Inc.	A07769
anti-human CD45-ECD	Beckman Coulter Inc.	A07784
anti-human CD56-PE/Cy5	Beckman Coulter Inc.	A07789
anti-human TCRαβ-PE	Beckman Coulter Inc.	A39499
anti-human TCR Vγ9-FITC	Beckman Coulter Inc.	IM1463
anti-human CD27-PE/Cy5	Beckman Coulter Inc.	6607107
anti-human CD45RA-ECD	Beckman Coulter Inc.	IM2711
anti-human CD69-PE	BD Biosciences	555531
anti-human NKG2D-PE	Beckman Coulter Inc.	A08934
Anti-humal IFNγ-PE	Beckman Coulter Inc.	IM2717U
Mouse IgG1 isotype control-PE	Beckman Coulter Inc.	A07796
Mouse IgG1 isotype control-ECD or PE/Cy5	Beckman Coulter Inc.	A07797A07798